Medical devices

ABSTRACT

Medical devices comprising an anti-connexin agent suitable for introduction into a subject.

This application is a National Stage Application under 35 U.S.C. §371 ofInternational Application No. PCT/US2008/014023, filed Dec. 22, 2008which claims the benefit of priority to U.S. Provisional Application No.61/008,738 filed on Dec. 21, 2007. The disclosures if both areincorporated herein by reference.

FIELD

The subject matter pertains to medical devices, including devices havinga composition, surface, or feature that exposes and/or delivers one ormore anti-connexin agents, with or without one or more other therapeuticagents and/or wound healing agents to tissue and/or fluid with whichthey come into contact on use or application.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art, or relevant, to thepresently described or claimed invention, or that any publication ordocument that is specifically or implicitly identified is prior art or areference that may be used in evaluating patentability. All documentsand other information referred to in this patent are incorporated hereinby reference in their entirety.

Over the past two decades, much research effort has been directedtowards the development of medical devices and machines that are used ina wide variety of clinical settings to repair, maintain, or enhancevital physiological functions of a subject mammal. For example, devicessuch as catheters, prosthetic heart valves, pacemakers, pulsegenerators, cardiac defibrillators, arteriovenous shunts, and stents areused extensively in the treatment of cardiac and other diseases. Otherexamples of medical devices, including screws, anchors, plates, staples,tacks, joints and similar devices, for example, are used in orthopedicsurgery. These implantable medical devices are made from a wide varietyof materials, including, for example, metals, plastics, and variouspolymeric materials. Other orthopedic devices include implants, such asimplants for hip, shoulder, elbow and knee replacements and surgeries,or craniomaxillofacial reconstruction, and implant coatings, as welldevices used in arthroscopic and laproscopic procedures, includingburrs, suture-passing instruments, arthroscopic shavers. Other examplesof medical devices include ocular devices, such as implants, includingintraocular lenses and glaucoma shunts. Still other devices includegastrointestinal implants. Other examples of medical devices includedevices for endoscopy, hysteroscopy, cytoscopy, bronchoscopy, etc.

When medical devices, for example, implantable medical devices, arebrought into contact with a subject, natural bodily processes can resultin inflammation, swelling, hypercellularity or other aberrant cellulardisposition, growth and/or proliferation, and/or tissue damage. Forexample, platelets may attach to the medical device which can result infurther complications at the site of use or implantation such as, forexample, thrombosis, leukocyte attachment, and/or neutrophil and/ormacrophage migration that lead to inflammation and/or aberrant cellulargrowth. An example of such a process resulting from the contacting of amedical device with a subject is provided by the use of a catheter orstent, which can result in tissue damage and/or restenosis. Restenosis,the reclosure of a peripheral or coronary artery following trauma tothat artery generally caused by efforts to open a stenosed or occludedportion of the artery, and resulting trauma may be caused by, forexample, balloon dilation, ablation, atherectomy or laser treatment ofthe artery. For example, balloon arterial injury reportedly results inendothelial denudation and subsequent regrowth of dysfunctionalendothelium that may contribute to the local smooth muscle cellproliferation and extracellular matrix production that result inreocclusion of the arterial lumen. Restenosis has been reported to occurin as many as 50% of patients undergoing such angioplasty procedures.Restenosis is believed to be a natural healing process in reaction tothe injury of the arterior wall caused by such angioplasty procedures.The healing process begins with the thrombotic mechanism at the site ofthe injury. The final steps of the healing process can be intimalhyperplasia, the uncontrolled migration and proliferation of medialsmooth muscle cells, combined with their extracellular matrixproduction, until the artery is again stenosed or occluded.

In humans and other mammals wound injury triggers an organized complexcascade of cellular and biochemical events that will in most casesresult in a healed wound. An ideally healed wound is one that restoresnormal anatomical structure, function, and appearance at the cellular,tissue, organ, and organism levels. Wound healing, whether initiated bytrauma, microbes or foreign materials, proceeds via a complex processencompassing a number of overlapping phases, including inflammation,epithelialization, angiogenesis and matrix deposition. Normally, theseprocesses lead to a mature wound and a certain degree of scar formation.Although inflammation and repair mostly occur along a prescribed course,the sensitivity of the process is dependent on the balance of a varietyof wound healing modulating factors, including for example, a network ofregulatory cytokines and growth factors.

Despite advances in the understanding of the principles underlying thewound healing process, there remains a significant unmet need insuitable therapeutic options for wound care, including delayed orcompromised wound healing of wounds such as chronic wounds, as well asswelling, inflammation, epithelialization rates and scarring associatedwith these and other wounds, including acute and subacute wounds.

Gap junctions are cell membrane structures that facilitate directcell-cell communication. A gap junction channel is formed of twoconnexons (hemichannels), each composed of six connexin subunits. Eachhexameric connexon docks with a connexon in the opposing membrane toform a single gap junction. Gap junction channels are reported to befound throughout the body. Tissue such as the corneal epithelium, forexample, has six to eight cell layers, yet is reported to expressesdifferent gap junction channels in different layers with connexin 43 inthe basal layer and connexin 26 from the basal to middle wing celllayers. In general, connexins are a family of proteins, commonly namedaccording to their molecular weight or classified on a phylogeneticbasis into alpha, beta, and gamma subclasses. At least 20 human and 19murine isoforms have been identified. Different tissues and cell typesare reported to have characteristic patterns of connexin proteinexpression and tissues such as cornea have been shown to alter connexinprotein expression pattern following injury or transplantation (Qui, C.et al., (2003) Current Biology, 13:1967-1703; Brander et al., (2004), J.Invest Dermatol. 122:1310-20).

It has been reported that, abnormal connexin function may be linked tocertain disease states (e.g. heart diseases) (A. C. de Carvalho, et al.,J Cardiovasc Electrophysiol 1994, 5 686). In certain connexin proteins,alterations in the turnover and trafficking properties may be induced bythe addition exogenous agents which may affect the level of gapjunctional intercellular communication (Darrow, B. J., et al. (1995).Circ Res 76: 381; Lin R, et al. (2001) J Cell Biol 154(4):815).Antisense technology has been reported for the modulation of theexpression for genes implicated in viral, fungal and metabolic diseases.See, e.g., U.S. Pat. No. 5,166,195, (oligonucleotide inhibitors of HIV),U.S. Pat. No. 5,004,810 (oligomers for hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting replication). See also U.S. Pat. No.7,098,190 to Becker et al. (formulations comprising antisensenucleotides to connexins). See also Becker and Green PCT/US06/04131(“Anti-connexin compounds and uses thereof').

There is a need for medical devices that will aid in ameliorating tissuedamage and/or enhancing tissue repair and/or limiting or inhibitingcomplications associated with using or implanting medical devices in asubject, such as for example, inflammation, restenosis and so on. Itwould be desirable to provide a medical device that aids in prevention,amelioration or treatment of” damaged tissue and/or inflammation, and/orthe enhancement of tissue repair, as well as swelling, hypercellularityor other aberrant cellular disposition, growth and/or proliferation,and/or tissue damage. Such devices, methods of manufacture, and usesthereof, are provided herein.

SUMMARY

The inventions described and claimed herein have many attributes andembodiments including, but not limited to, those set forth or describedor referenced in this Summary. The inventions described and claimedherein are not limited to or by the features or embodiments identifiedin this Summary, which is included for purposes of illustration only andnot restriction.

The present invention is in part directed to new medical devices whichmay be used, for example, in the treatment and prevention of variousdiseases, disorders and/or conditions, including diseases, disorders,and/or conditions relating to tissue damage and/or inflammation, whetheror not in excess, in a mammal including, but not limited to, the kindreferenced herein, and/or enhancing tissue repair processes and/orameliorating tissue damage and/or inflammation.

In one aspect, the invention also relates to a medical device for use orimplantation in a subject, wherein said medical device comprises one ormore anti-connexin agents in combination with one or more therapeuticagents, one or more gap junction modulating agents, and/or one or morewound healing agents.

In another aspect, the invention relates a medical device for use orimplantation in a subject, wherein said medical device comprises ananti-connexin agent. In one embodiment, the anti-connexin agent is ananti-connexin 43 agent.

In another aspect, the invention relates a medical device for use orimplantation in a subject, wherein said medical device comprises one ormore anti-connexin polynucleotides.

In another aspect, the invention relates a medical device for use orimplantation in a subject, wherein said medical device comprises one ormore anti-connexin polypeptides.

In another aspect, the invention relates a medical device for use orimplantation in a subject, wherein said medical device comprises one ormore gap junction modulating agents.

In another aspect, the invention relates a medical device for use orimplantation in a subject, wherein said medical device comprises ananti-connexin polypeptide and an anti-connexin polynucleotide.

In another aspect, the invention relates a medical device for use orimplantation in a subject, wherein said medical device comprises a firstcoating containing one or more anti-connexin polynucleotides and asecond coating containing one or more anti-connexin polypeptides wherebyupon implantation or application of said device in a subject theanti-connexin polypeptide and the anti-connexin polynucleotide aresequentially administered. Preferably the anti-connexin polypeptide isadministered first. In one embodiment, the medical device contains acontrolled release formulation whereby the anti-connexin polypeptide andthe anti-connexin polynucleotide are sequentially released.

In another aspect, the invention relates a medical device for use orimplantation in a subject, wherein said medical device comprises one ormore anti-connexin polynucleotides, one or more anti-connexinpolypeptides, one or more therapeutic agents, one or more gap junctionmodifying agents, and/or one or more agents useful for wound healing. Infurther embodiments, the medical device contains layers foradministration, portions of the medical device coated with differentsubstances released at different rates, a controlled release formulationor mechanics to facilitate administration of said combination in anyorder, or means for mechanical administration in any desired order.

In one aspect, the invention relates to an implantable medical devicecomprising an anti-connexin agent releasable upon insertion of themedical device to or within a subject.

In one aspect, a medical device for use or implantation in a mammal,comprising an anti-connexin 43 compound releasable upon insertion of themedical device to or within a subject, is provided. In one embodiment,the anti-connexin 43 compound decreases connexin 43 protein expression.In another embodiment, the anti-connexin 43 compound is an antisenseoligonucleotide. In another embodiment, the anti-connexin 43 compound isa siRNA oligonucleotide. In another embodiment, the anti-connexin 43compound is an RNAi oligonucleotide. In another embodiment, theanti-connexin 43 compound is a peptide compound, e.g., a peptide thatblocks or inhibits hemichannel opening. In another embodiment, theanti-connexin 43 compound is a peptidomimetic, e.g., a peptidomimeticthat blocks or inhibits hemichannel opening. In another embodiment, theanti-connexin 43 compound is an anti-connexin 43 antibody or antigenbinding fragment thereof. In another embodiment, the anti-connexin 43compound is a monoclonal antibody. In another embodiment, theanti-connexin 43 compound is an F(v), Fab, Fab′ or F(ab′)₂ anti-connexin43 antibody fragment. In another embodiment, the anti-connexin 43compound is a chimeric or humanized antibody. In a further embodiment,the antibody fragment is a chimeric or humanized antibody fragment. Inanother embodiment, the anti-connexin 43 compound binds to connexin 43mRNA. In another embodiment, the anti-connexin 43 compound binds to aconnexin 43 hemichannel. In another embodiment, the anti-connexin 43compound binds to a hemichannel extracellular loop. In anotherembodiment, the anti-connexin 43 compound blocks or inhibits a connexin43 hemichannel. In another one embodiment, the anti-connexin 43 compoundblocks or inhibits connexin 43 hemichannel opening. In anotherembodiment, the anti-connexin 43 compound is in a polynucleotide havinga sequence selected for SEQ.ID.NOS.1 or 2 or a pharmaceuticallyacceptable salt thereof. In another embodiment, the anti-connexin 43compound is a peptide having a sequence selected from SEQ.ID.NOS. 15 to38 or a pharmaceutically acceptable salt thereof. In another embodiment,the anti-connexin 43 compound is selected from the group consisting ofan antisense oligonucleotides, siRNA, and RNAi. In another embodiment,the anti-connexin 43 compound is a peptide of SEQ.ID.NOS. 18 or 19. Inanother embodiment, the anti-connexin 43 compound is a peptide ofSEQ.ID.NO.19. In another embodiment, the anti-connexin 43 compound is apolynucleotide having a sequence selected from the group consisting of:SEQ.ID.NOS. 1 to 12. In another embodiment, the surface of the medicaldevice comprises an anti-connexin or anti-connexin 43 agent. In anotherembodiment, the anti-connexin or anti-connexin 43 agent is present at aweight percentage of about 0.0001% to about 30%.

In another embodiment, the surface of the medical device contacts atarget tissue within the subject upon use. In another embodiment, thetarget tissue is heart tissue or vascular tissue, or other tissueexposed to a device during or after a surgical procedure. In anotherembodiment, the release rate of the anti-connexin agent is controlled.In another embodiment, the medical device provides for surface contactrelease of the anti-connexin agent. In another embodiment, the medicaldevice provides for the sustained release of the anti-connexin agent. Inanother embodiment, the medical device provides for the slow release ofthe anti-connexin agent. In another embodiment, the device comprises acoating containing an anti-connexin agent. In a further embodiment, thecoating comprises a polymer. In another embodiment, the coatingcomprises a plurality of layers of a polymer/anti-connexin agentmixture. In another embodiment, the medical device is for implantationin a human. In another embodiment, the medical device comprises a stent.In another embodiment, the stent is a drug-eluting stent. In anotherembodiment, the medical device comprises a balloon, a prosthetic heartvalve, an annuloplasty ring, a pulse generator, a cardiac defibrillator,an arteriovenous shunt, an anastomosis device, a hemostatic barrier or apacemaker. In another embodiment, the medical device comprises anorbital implant, a lens, a lens implant, a corneal implant, or aglaucoma shunt. In another embodiment, the medical device comprises anorthopedic plate, a bone pin, a bone substitute, an anchor, a joint, ascrew, or a vertebral disk. In another embodiment, the medical devicecomprises a device for knee, elbow, shoulder, or hip replacement, inwhole or in part. In another embodiment, the medical device comprises agastrointestinal implant. In another embodiment, the medical device isan endoscopy device. In another embodiment, the medical device comprisesa graft, a shunt, a vascular implant, a tissue scaffold, an intraluminaldevice or a vascular support. In other embodiments, the medical deviceis for implantation in a non-human animal or bird. In anotherembodiment, the medical device comprises at least one channel formed inan outer surface thereof, and wherein the anti-connexin agent isincluded on and/or within at least one channel. In another embodiment,at least a portion of the medical device is formed, in whole or in part,of a substance that includes the anti-connexin agent. In anotherembodiment, the device further comprises one or more therapeutic agents.In another embodiment, the medical device is for use in a human ornon-human animal or bird.

In another aspect, a method of preventing and/or treating damageassociated with the use or implantation of a medical device in a subjectcomprising introducing into the subject a medical device of which atleast a portion comprises an anti-connexin agent, wherein the damage isprevented, amerliorated and/or delayed, is provided. In one embodiment,the subject is a mammal. In one embodiment, the mammal is a human. Inone embodiment, the mammal is selected from the group consisting ofdomestic and pet animals, sports animals, farm animals, zoo animals, andbirds. In one embodiment, the mammal is a horse, a dog, or a cat.

In yet another aspect, a method of preventing and/or treating damageassociated with the use or implantation of a medical device in a subjectcomprising use or implantation of a medical device which comprises ananti-connexin agent that is releasable at its point of contact, whereinthe damage is prevented, amerliorated and/or reduced, is provided.

DETAILED DESCRIPTION Definitions

Before further describing the inventions in general and in terms ofvarious nonlimiting specific embodiments, certain terms used in thecontext of the describing the invention are set forth. Unless indicatedotherwise, the following terms have the following meanings when usedherein and in the appended claims. Those terms that are not definedbelow or elsewhere in the specification shall have their art-recognizedmeaning.

As used herein, “subject” refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, horses, cats, sheep, pigs, cows, etc. Thepreferred subject is a human.

As used herein, “mammal” refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, or petanimals, such as dogs, horses, cats, sheep, pigs, cows, etc. Thepreferred mammal herein is a human, including adults, children, and theelderly.

As used herein, a “disorder” is any disorder, disease, or condition thatwould benefit from an agent that reduces damage associated with the useor implantation of a medical device. Disorders include, but are notlimited to, damage to tissue (including, e.g., heart, liver, kidney, CNS(including brain), joint, gastrointestinal tissue) and vascular damage.

As used herein, “pharmaceutically acceptable salts” refers to saltsprepared from pharmaceutically acceptable non-toxic bases or acidsincluding inorganic or organic bases and inorganic or organic acids thelike. When the anti-connexin agent compound is basic, salts may beprepared from pharmaceutically acceptable non-toxic acids, includinginorganic and organic acids. Such acids include acetic, benzenesulfonic,benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic,glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic,mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, andthe like

As used herein, “preventing” or “prevention” means preventing in wholeor in part, ameliorating or controlling in whole or in part, orreducing, decreasing, lessening or retarding in whole or in part.

The term “therapeutically effective amount” or “effective amount” meansthe amount of the subject compound that will elicit a desired response,for example, a biological or medical result or response of a tissue,system, animal or human that is sought, for example, by a researcher,veterinarian, medical doctor, or other clinician. That result can bealleviation of the signs, symptoms, or causes of a disease or disorderor condition, or any other desired alteration of a biological system. Inthe present invention, the result will typically involve, for example,the prevention, decrease, or reversal of tissue injury or damage,including inflammation, in whole or in part, associated with the use orimplantation (temporary or permanent) of a medical device. The resultwill also typically involve the prevention, decrease, or reversal ofinflammation.

As used herein, the term “treatment” or “treating” refers to boththerapeutic treatment and prophylactic or preventative measures.Prevention or prophylaxis may be in whole or in part.

The term “damage associated with the use or implantation of a medicaldevice” refers to damage or injury to tissue resulting from theinsertion, presence, manipulation or removal of a medical device.“Damage associated with the use or implantation of a medical device” maybe evidenced by one or more conditions, including an inflammatoryresponse, a proliferative response including neointimal proliferation,cellular proliferation, removal of endothelium and damage to smoothmuscle cells, stenosis or restenosis following angioplasty or insertionand removal of a medical device, neointimal hyperplasia leading tostenosis or restenosis, platelet adhesion restenosis, and otherinflammatory processes which are associated with implantation, use andremoval of a medical device, including swelling.

The terms “peptidomimetic” and “mimetic” include naturally occurring andsynthetic chemical compounds that may have substantially the samestructural and functional characteristics of protein regions which theymimic. In the case of connexins, these may mimic, for example, theextracellular loops of opposing connexins involved in connexon-connexondocking and cell-cell channel formation, and/or the extracellular loopsof hemichannel connexins.

As used herein, the term “peptide analogs” refer to the compounds withproperties analogous to those of the template peptide and can benon-peptide drugs. “Peptidomimetics” (also known as peptide mimetics)which include peptide-based compounds, also include such non-peptidebased compounds such as peptide analogs. Peptidomimetics that arestructurally similar to therapeutically useful peptides can be used toproduce an equivalent or enhanced therapeutic or prophylactic effect.Generally, peptidomimetics are structural or functional mimics (e.g.identical or similar) to a paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological function or activity), but canalso have one or more peptide linkages optionally replaced by a linkageselected from the group consisting of, for example, —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—.The mimetic can be either entirely composed of natural amino acids,synthetic chemical compounds, non-natural analogues of amino acids, or,is a chimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also comprise anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter mimetic activity. In thecase of connexins, these can mimic, for example, the extracellular loopsof opposing connexins involved in connexon-connexon docking andcell-cell channel formation. For example, a mimetic composition can beuseful as a gap junction modulating agent if it is capable ofdown-regulating biological actions or activities of connexons, such as,for example, preventing the docking of connexons to formgap-junction-mediated cell-cell communications, or preventing theopening of connexons to expose the cell cytoplasm to the extracellularmillieu. Peptidomimetics encompass those described herein, as well asthose as may be known in the art, whether now known or later developed.

The term “composition” is intended to encompass a product comprising oneor more ingredients.

In general, the terms “modulator” and “modulation” of gap junctionactivity, as used herein in its various forms, refers to inhibition inwhole or in part of the action or activity of a gap junction or ahemichannel and may function as gap junction modulating agents.

In general, the term “protein” refers to any polymer of two or moreindividual amino acids (whether or not naturally occurring) linked viapeptide bonds, as occur when the carboxyl carbon atom of the carboxylicacid group bonded to the alpha-carbon of one amino acid (or amino acidresidue) becomes covalently bound to the amino nitrogen atom of theamino group bonded to the alpha-carbon of an adjacent amino acid. Thesepeptide bond linkages, and the atoms comprising them (i.e., alpha-carbonatoms, carboxyl carbon atoms (and their substituent oxygen atoms), andamino nitrogen atoms (and their substituent hydrogen atoms)) form the“polypeptide backbone” of the protein. In addition, as used herein, theterm “protein” is understood to include the terms “polypeptide” and“peptide” (which, at times, may be used interchangeably herein).Similarly, protein fragments, analogs, derivatives, and variants are maybe referred to herein as “proteins,” and shall be deemed to be a“protein” unless otherwise indicated. The term “fragment” of a proteinrefers to a polypeptide comprising fewer than all of the amino acidresidues of the protein. A “domain” of a protein is also a fragment, andcomprises the amino acid residues of the protein often required toconfer activity or function.

As used herein, it is to be understood that reference to a particularconnexin is a reference to all species variants thereof, even if theirmolecular weights are different. Thus, for example, a reference to“connexin 43” means not only human connexin 43 but to the analogousconnexin in each other species, no matter whether they are also 43 Kd.Similarly, reference to a non-human “connexin 43” is a reference to theconnexin 43 analog or variant in that species. Thus, for example,reference to “horse connexin 43” is a reference to the relevant analogor variant of human connexin 43 in horse even if it does not have a 43Kd molecular weight.

Anti-Connexin Agents

Anti-connexin agents of the invention described herein are capable ofmodulating or affecting the transport of molecules into and out of cells(e.g., blocking or inhibiting or downregulating). Thus certainanti-connexin agents described herein modulate cellular communication(e.g., cell to cell). Certain anti-connexin agents modulate or effecttransmission of molecules between the cell cytoplasm and the periplasmicor extracellular space. Such anti-connexin agents are generally targetedto connexins and/or connexin hemichannels (connexons). Hemichannels andresulting gap junctions that comprise connexins are independentlyinvolved in the release or exchange of small molecules between the cellcytoplasm and an extracellular space or tissue in the case of openhemichannels, and between the cytoplasm of adjoining cell in the case ofopen gap junctions. Thus, an anti-connexin agents provided herein maydirectly or indirectly reduce coupling and communication between cellsor reduce or block communication (or the transmission of molecules)between a cell and extracellular space or tissue, and the modulation oftransport of molecules from a cell into an extracellular space or tissue(or from an extracellular space or tissue into a cell) or betweenadjoining cells is within the scope of anti-connexin agents andembodiments of the invention.

Any anti-connexin agent that is capable of eliciting a desiredinhibition of the passage (e.g. transport) of molecules through a gapjunction or connexin hemichannel may be used in embodiments of theinvention. Any anti-connexin agents that modulates the passage ofmolecules through a gap junction or connexin hemichannel are alsoprovided in particular embodiments (e.g., those that modulate, block orlessen the passage of molecules from the cytoplasm of a cell into anextracellular space or adjoining cell cytoplasm). Such anti-connexinagents may modulate the passage of molecules through a gap junction orconnexin hemichannel with or without gap junction uncoupling (blockingthe transport of molecules through gap junctions). Such compoundsinclude, for example, proteins and polypeptides, polynucleotides, andother organic compounds, and they may, for example block the function orexpression of a gap junction or a hemichannel in whole or in part, ordownregulate the production of a connexin in whole or in part. Certaingap junction inhibitors are listed in Evans, W. H. and Boitano, S.Biochem. Soc. Trans. 29: 606-612 (2001).

Certain anti-connexin agents provide downregulation or inhibition ofconnexin expression (for example, by downregulation of mRNAtranscription or translation) or otherwise decrease or inhibit theactivity of a connexin protein, a connexin hemichannel or a gapjunction. In the case of downregulation, this will have the effect ofreducing direct cell-cell communication by gap junctions, or exposure ofcell cytoplasm to the extracellular space by hemichannels, at the siteat which connexin expression is down-regulated.

Examples of anti-connexin agents include agents that decrease or inhibitexpression or function of connexin mRNA and/or protein or that decreaseactivity, expression or formation of a connexin, a connexin hemichannelor a gap junction. Anti-connexin agents include anti-connexinpolynucleotides, such as antisense polynucleotides and otherpolynucleotides (such as polynucleotides having siRNA or ribozymefunctionalities), as well as antibodies and binding fragments thereof,and peptides and polypeptides, including peptidomimetics and peptideanalogs that modulate hemichannel or gap junction activity or function.

Anti-Connexin Polynucleotides

Anti-connexin polynucleotides include connexin antisense polynucleotidesas well as polynucleotides which have functionalities which enable themto downregulate connexin expression. Other suitable anti-connexinpolynucleotides include RNAi polynucleotides and siRNA polynucleotides.

Synthesis of antisense polynucleotides and other anti-connexinpolynucleotides such as RNAi, siRNA, and ribozyme polynucleotides aswell as polynucleotides having modified and mixed backbones is known tothose of skill in the art. See e.g. Stein C. A. and Krieg A. M. (eds),Applied Antisense Oligonucleotide Technology, 1998 (Wiley-Liss). Methodsof synthesizing antibodies and binding fragments as well as peptides andpolypeptides, including peptidomimetics and peptide analogs are known tothose of skill in the art. See e.g. Lihu Yang et al., Proc. Natl. Acad.Sci. U.S.A., 1; 95(18): 10836-10841 (Sep. 1, 1998); Harlow and Lane(1988) “Antibodies: A Laboratory Manuel” Cold Spring HarborPublications, New York; Harlow and Lane (1999) “Using Antibodies” ALaboratory Manuel, Cold Spring Harbor Publications, New York.

According to one aspect, the downregulation of connexin expression maybe based generally upon the antisense approach using antisensepolynucleotides (such as DNA or RNA polynucleotides), and moreparticularly upon the use of antisense oligodeoxynucleotides (ODN).These polynucleotides (e.g., ODN) target the connexin protein (s) to bedownregulated. Typically the polynucleotides are single stranded, butmay be double stranded.

The antisense polynucleotide may inhibit transcription and/ortranslation of a connexin. Preferably the polynucleotide is a specificinhibitor of transcription and/or translation from the connexin gene ormRNA, and does not inhibit transcription and/or translation from othergenes or mRNAs. The product may bind to the connexin gene or mRNA either(i) 5′ to the coding sequence, and/or (ii) to the coding sequence,and/or (iii) 3′ to the coding sequence.

The antisense polynucleotide is generally antisense to a connexin mRNA.Such a polynucleotide may be capable of hybridizing to the connexin mRNAand may thus inhibit the expression of connexin by interfering with oneor more aspects of connexin mRNA metabolism including transcription,mRNA processing, mRNA transport from the nucleus, translation or mRNAdegradation. The antisense polynucleotide typically hybridizes to theconnexin mRNA to form a duplex which can cause direct inhibition oftranslation and/or destabilization of the mRNA. Such a duplex may besusceptible to degradation by nucleases.

The antisense polynucleotide may hybridize to all or part of theconnexin mRNA. Typically the antisense polynucleotide hybridizes to theribosome binding region or the coding region of the connexin mRNA. Thepolynucleotide may be complementary to all of or a region of theconnexin mRNA. For example, the polynucleotide may be the exactcomplement of all or a part of connexin mRNA. However, absolutecomplementarity is not required and polynucleotides which havesufficient complementarity to form a duplex having a melting temperatureof greater than about 20° C., 30° C. or 40° C. under physiologicalconditions are particularly suitable for use in the present invention.

Thus the polynucleotide is typically a homologue of a sequencecomplementary to the mRNA. The polynucleotide may be a polynucleotidewhich hybridizes to the connexin mRNA under conditions of medium to highstringency such as 0.03M sodium chloride and 0.03M sodium citrate atfrom about 50° C. to about 60° C.

For certain aspects, suitable polynucleotides are typically from about 6to 40 nucleotides in length. Preferably a polynucleotide may be fromabout 12 to about 35 nucleotides in length, or alternatively from about12 to about 20 nucleotides in length or more preferably from about 18 toabout 32 nucleotides in length. According to an alternative aspect, thepolynucleotide may be at least about 40, for example at least about 60or at least about 80, nucleotides in length and up to about 100, about200, about 300, about 400, about 500, about 1000, about 2000 or about3000 or more nucleotides in length.

The connexin protein or proteins targeted by the polynucleotide will bedependent upon the site at which downregulation is to be effected. Thisreflects the non-uniform make-up of gap junction(s) at different sitesthroughout the body in terms of connexin sub-unit composition. Theconnexin is a connexin that naturally occurs in a human or animal in oneaspect or naturally occurs in the tissue in which connexin expression oractivity is to be decreased. The connexin gene (including codingsequence) generally has homology with the coding sequence of one or moreof the specific connexins mentioned herein, such as homology with theconnexin 43 coding sequence shown in Table 8. The connexin is typicallyan α or β connexin. Preferably the connexin is an α connexin and isexpressed in the tissue to be treated.

Some connexin proteins are however more ubiquitous than others in termsof distribution in tissue. One of the most widespread is connexin 43.Polynucleotides targeted to connexin 43 are particularly suitable foruse in the present invention. In other aspects other connexins aretargeted.

Anti-connexin polynucleotides include connexin antisense polynucleotidesas well as polynucleotides which have functionalities which enable themto downregulate connexin expression. Other suitable anti-connexinpolynucleotides include RNAi polynucleotides and SiRNA polynucleotides.

In one preferred aspect, the antisense polynucleotides are targeted tothe mRNA of one connexin protein only. Most preferably, this connexinprotein is connexin 43. In another aspect, connexin protein is connexin26, 30, 31.1, 32, 36, 37, 40, or 45. In other aspects, the connexinprotein is connexin 30.3, 31, 40.1, or 46.6.

It is also contemplated that polynucleotides targeted to separateconnexin proteins be used in combination (for example 1, 2, 3, 4 or moredifferent connexins may be targeted). For example, polynucleotidestargeted to connexin 43, and one or more other members of the connexinfamily (such as connexin 26, 30, 30.3, 31.1, 32, 36, 37, 40, 40.1, 45,and 46.6) can be used in combination.

Alternatively, the antisense polynucleotides may be part of compositionswhich may comprise polynucleotides to more than one connexin protein.Preferably, one of the connexin proteins to which polynucleotides aredirected is connexin 43. Other connexin proteins to whicholigodeoxynucleotides are directed may include, for example, connexins26, 30, 30.3, 31.1, 32, 36, 37, 40, 40.1, 45, and 46.6. Suitableexemplary polynucleotides (and ODNs) directed to various connexins areset forth in Table 1.

Individual antisense polynucleotides may be specific to a particularconnexin, or may target 1, 2, 3 or more different connexins. Specificpolynucleotides will generally target sequences in the connexin gene ormRNA which are not conserved between connexins, whereas non-specificpolynucleotides will target conserved sequences for various connexins.

The polynucleotides for use in the invention may suitably be unmodifiedphosphodiester oligomers. Such oligodeoxynucleotides may vary in length.A 30 mer polynucleotide has been found to be particularly suitable.

Many aspects of the invention are described with reference tooligodeoxynucleotides. However it is understood that other suitablepolynucleotides (such as RNA polynucleotides) may be used in theseaspects.

The antisense polynucleotides may be chemically modified. This mayenhance their resistance to nucleases and may enhance their ability toenter cells. For example, phosphorothioate oligonucleotides may be used.Other deoxynucleotide analogs include methylphosphonates,phosphoramidates, phosphorodithioates, N3′P5′-phosphoramidates andoligoribonucleotide phosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates. Alternatively mixedbackbone oligonucleotides (“MBOs”) may be used. MBOs contain segments ofphosphothioate oligodeoxynucleotides and appropriately placed segmentsof modified oligodeoxy- or oligoribonucleotides. MBOs have segments ofphosphorothioate linkages and other segments of other modifiedoligonucleotides, such as methylphosphonate, which is non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides. Methodsof preparing modified backbone and mixed backbone oligonucleotides areknown in the art.

The precise sequence of the antisense polynucleotide used in theinvention will depend upon the target connexin protein. In oneembodiment, suitable connexin antisense polynucleotides can includepolynucleotides such as oligodeoxynucleotides selected from thefollowing sequences set forth in Table 1:

TABLE 1 5′ GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC 3′ (connexin 43)(SEQ. ID. NO: 1) 5′ GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC 3′(connexin 43) (SEQ. ID. NO: 2) 5′GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT 3′ (connexin 43)(SEQ. ID. NO: 3) 5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA 3′ (connexin 26)(SEQ. ID. NO: 4) 5′ CAT CTC CTT GGT GCT CAA CC 3′ (connexin 37)(SEQ. ID. NO: 5) 5′ CTG AAG TCG ACT TGG CTT GG 3′ (connexin 37)(SEQ. ID. NO: 6) 5′ CTC AGA TAG TGG CCA GAA TGC 3′ (connexin 30)(SEQ. ID. NO: 7) 5′ TTG TCC AGG TGA CTC CAA GG 3′ (connexin 30)(SEQ. ID. NO: 8) 5′ CGT CCG AGC CCA GAA AGA TGA GGT C 3′ (connexin 31.1)(SEQ. ID. NO: 9) 5′ AGA GGC GCA CGT GAG ACA C 3′ (connexin 31.1)(SEQ. ID. NO: 10) 5′ TGA AGA CAA TGA AGA TGT T 3′ (connexin 31.1)(SEQ. ID. NO: 11) 5′ TTT CTT TTC TAT GTG CTG TTG GTG A 3′ (connexin 32)(SEQ. ID. NO: 12)

Suitable polynucleotides for the preparation of the combinedpolynucleotide compositions described herein include for example,polynucleotides to Connexin Cx43 and polynucleotides for connexins 26,30, 31.1, 32 and 37 as described in Table 1 above.

Although the precise sequence of the antisense polynucleotide used inthe invention will depend upon the target connexin protein, for connexin43, antisense polynucleotides having the following sequences have beenfound to be particularly suitable:

(SEQ. ID. NO: 1) GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC;(SEQ. ID. NO: 2) GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC; and(SEQ. ID. NO: 3) GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT.

For example, suitable antisense polynucleotides for connexins 26, 31.1and 32 have the following sequences:

(SEQ. ID. NO: 4) 5′ TCC TGA GCA ATA CCT AAC GAA CAA ATA (connexin 26);(SEQ. ID. NO: 9) 5′ CGT CCG AGC CCA GAA AGA TGA GGT C (connexin 31.1);and (SEQ. ID. NO: 12) 5′ TTT CTT TTC TAT GTG CTG TTG GTG A(connexin 32).

Other connexin antisense polynucleotide sequences useful according tothe methods of the present invention include:

(SEQ. ID. NO: 5) 5′ CAT CTC CTT GGT GCT CAA CC 3′ (connexin 37);(SEQ. ID. NO: 6) 5′ CTG AAG TCG ACT TGG CTT GG 3′ (connexin 37);(SEQ. ID. NO: 7) 5′ CTC AGA TAG TGG CCA GAA TGC 3′ (connexin 30);(SEQ. ID. NO: 8) 5′ TTG TCC AGG TGA CTC CAA GG 3′ (connexin 30);(SEQ. ID. NO: 10) 5′ AGA GGC GCA CGT GAG ACA C 3′ (connexin 31.1); and(SEQ. ID. NO: 11) 5′ TGA AGA CAA TGA AGA TGT T 3′ (connexin 31.1).

Polynucleotides, including ODN's, directed to connexin proteins can beselected in terms of their nucleotide sequence by any convenient, andconventional, approach. For example, the computer programs MacVector andOligoTech (from Oligos etc. Eugene, Oreg., USA) can be used. Onceselected, the ODN's can be synthesized using a DNA synthesizer.

Polynucleotide Homologues

Homology and homologues are discussed herein (for example, thepolynucleotide may be a homologue of a complement to a sequence inconnexin mRNA). Such a polynucleotide typically has at least about 70%homology, preferably at least about 80%, at least about 90%, at leastabout 95%, at least about 97% or at least about 99% homology with therelevant sequence, for example over a region of at least about 15, atleast about 20, at least about 40, at least about 100 more contiguousnucleotides (of the homologous sequence).

Homology may be calculated based on any method in the art. For examplethe UWGCG Package provides the BESTFIT program which can be used tocalculate homology (for example used on its default settings) (Devereuxet al (1984) Nucleic Acids Research 12, p 387-395). The PILEUP and BLASTalgorithms can be used to calculate homology or line up sequences(typically on their default settings), for example as described inAltschul S. F. (1993) J Mol Evol 36: 290-300; Altschul, S, F et al(1990) J Mol Biol 215: 403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached.

The BLAST algorithm parameters W, T and X determine the sensitivity andspeed of the alignment. The BLAST program uses as defaults a word length(W), the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to a second sequence isless than about 1, preferably less than about 0.1, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The homologous sequence typically differs from the relevant sequence byat least about (or by no more than about) 2, 5, 10, 15, 20 moremutations (which may be substitutions, deletions or insertions). Thesemutations may be measured across any of the regions mentioned above inrelation to calculating homology.

The homologous sequence typically hybridizes selectively to the originalsequence at a level significantly above background. Selectivehybridization is typically achieved using conditions of medium to highstringency (for example 0.03M sodium chloride and 0.03M sodium citrateat from about 50° C. to about 60° C.). However, such hybridization maybe carried out under any suitable conditions known in the art (seeSambrook et al. (1989), Molecular Cloning: A Laboratory Manual). Forexample, if high stringency is required, suitable conditions include0.2×SSC at 60° C. If lower stringency is required, suitable conditionsinclude 2×SSC at 60° C.

Peptide and Polypeptide Anti-Connexin Agents

Binding proteins, including peptides, peptidomimetics, antibodies,antibody fragments, and the like, are also suitable modulators of gapjunctions and hemichannels.

Binding proteins include, for example, monoclonal antibodies, polyclonalantibodies, antibody fragments (including, for example, Fab, F(ab′)₂ andFv fragments; single chain antibodies; single chain Fvs; and singlechain binding molecules such as those comprising, for example, a bindingdomain, hinge, CH2 and CH3 domains, recombinant antibodies and antibodyfragments which are capable of binding an antigenic determinant (i.e.,that portion of a molecule, generally referred to as an epitope) thatmakes contact with a particular antibody or other binding molecule.These binding proteins, including antibodies, antibody fragments, and soon, may be chimeric or humanized or otherwise made to be lessimmunogenic in the subject to whom they are to be administered, and maybe synthesized, produced recombinantly, or produced in expressionlibraries. Any binding molecule known in the art or later discovered isenvisioned, such as those referenced herein and/or described in greaterdetail in the art. For example, binding proteins include not onlyantibodies, and the like, but also ligands, receptors, peptidomimetics,or other binding fragments or molecules (for example, produced by phagedisplay) that bind to a target (e.g. connexin, hemichannel, orassociated molecules).

Binding molecules will generally have a desired specificity, includingbut not limited to binding specificity, and desired affinity. Affinity,for example, may be a K_(a) of greater than or equal to about 10⁴ M⁻¹,greater than or equal to about 10⁶ M⁻¹, greater than or equal to about10⁷ M⁻¹, greater than or equal to about 10⁸ M⁻¹. Affinities of evengreater than about 10⁸ M⁻¹ are suitable, such as affinities equal to orgreater than about 10⁹ M⁻¹, about 10¹⁰ M⁻¹, about 10¹¹ M⁻¹, and about10¹² M⁻¹. Affinities of binding proteins according to the presentinvention can be readily determined using conventional techniques, forexample those described by Scatchard et al., 1949 Ann. N.Y. Acad. Sci.51: 660.

By using data obtained from hydropathy plots, it has been proposed thata connexin contains four-transmembrane-spanning regions and two shortextra-cellular loops. The positioning of the first and secondextracellular regions of connexin was further characterized by thereported production of anti-peptide antibodies used forimmunolocalization of the corresponding epitopes on split gap junctions.Goodenough D. A. J Cell Biol 107: 1817-1824 (1988); Meyer R. A., J CellBiol 119: 179-189 (1992).

The extracellular domains of a hemichannel contributed by two adjacentcells “dock” with each other to form complete gap junction channels.Reagents that interfere with the interactions of these extracellulardomains can impair cell-to-cell communication. Peptide inhibitors of gapjunctions and hemichannels have been reported. See for example Berthoud,V. M. et al., Am J. Physiol. Lung Cell Mol. Physiol. 279: L619-L622(2000); Evans, W. H. and Boitano, S. Biochem. Soc. Trans. 29: 606-612,and De Vriese A. S., et al. Kidney Int. 61: 177-185 (2001). Shortpeptides corresponding to sequences within the extracellular loops ofconnexins were said to inhibit intercellular communication. Boitano S,and Evans W. Am J Physiol Lung Cell Mol Physiol 279: L623-L630 (2000).The use of peptides as inhibitors of cell-cell channel formationproduced by connexin (Cx) 32 expressed in paired Xenopus oocytes hasalso been reported. Dahl G, et al., Biophys J 67: 1816-1822 (1994).Berthoud, V. M. and Seul, K. H., summarized some of these results. AmJ., Physiol. Lung Cell Mol. Physiol. 279: L619-L622 (2000).

Anti-connexin agents include peptides comprising an amino acid sequencecorresponding to a transmembrane region (e.g. 1^(st) to 4^(th)) of aconnexin (e.g. connexin 45, 43, 26, 30, 31.1, and 37). Anti-connexinagents may comprise a peptide comprising an amino acid sequencecorresponding to a portion of a transmembrane region of a connexin 45.Anti-connexin agents include a peptide having an amino acid sequencethat comprises about 5 to 20 contiguous amino acids of SEQ.ID.NO:13, apeptide having an amino acid sequence that comprises about 8 to 15contiguous amino acids of SEQ.ID.NO:13, or a peptide having an aminoacid sequence that comprises about 11 to 13 contiguous amino acids ofSEQ.ID.NO:13. Other embodiments are directed to an anti-connexin agentthat is a peptide having an amino acid sequence that comprises at leastabout 5, at least about 6, at least about 7, at least about 8, at leastabout 9, at least about 10, at least about 11, at least about 12, atleast about 13, at least about 14, at least about 15, at least about 20,at least about 25, or at least about 30 contiguous amino acids ofSEQ.ID.NO:13. In certain anti-connexin agents provided herein, theextracellular domains of connexin 45 corresponding to the amino acids atpositions 46-75 and 199-228 of SEQ ID NO:13 may be used to develop theparticular peptide sequences. Certain peptides described herein have anamino acid sequence corresponding to the regions at positions 46-75 and199-228 of SEQ.ID.NO: 13. The peptides need not have an amino acidsequence identical to those portions of SEQ.ID.NO: 13, and conservativeamino acid changes may be made such that the peptides retain bindingactivity or functional activity. Alternatively, the peptide may targetregions of the connexin protein other than the extracellular domains(e.g. the portions of SEQ.ID.NO:13 not corresponding to positions 46-75and 199-228).

Also, suitable anti-connexin agents comprise a peptide comprising anamino acid sequence corresponding to a portion of a transmembrane regionof a connexin 43. Anti-connexin agents include peptides having an aminoacid sequence that comprises about 5 to 20 contiguous amino acids ofSEQ.ID.NO:14, peptides having an amino acid sequence that comprisesabout 8 to 15 contiguous amino acids of SEQ.ID.NO:14, or peptides havingan amino acid sequence that comprises about 11 to 13 contiguous aminoacids of SEQ.ID.NO:14. Other anti-connexin agents include a peptidehaving an amino acid sequence that comprises at least about 5, at leastabout 6, at least about 7, at least about 8, at least about 9, at leastabout 10, at least about 11, at least about 12, at least about 13, atleast about 14, at least about 15, at least about 20, at least about 25,or at least about 30 contiguous amino acids of SEQ.ID.NO:14. Otheranti-connexin agents comprise the extracellular domains of connexin 43corresponding to the amino acids at positions 37-76 and 178-208 ofSEQ.ID.NO: 14. Anti-connexin agents include peptides described hereinwhich have an amino acid sequence corresponding to the regions atpositions 37-76 and 178-208 of SEQ.ID.NO: 14. The peptides need not havean amino acid sequence identical to those portions of SEQ.ID.NO: 14, andconservative amino acid changes may be made such that the peptidesretain binding activity or functional activity. Alternatively, peptidesmay target regions of the connexin protein other than the extracellulardomains (e.g. the portions of SEQ.ID.NO:14 not corresponding topositions 37-76 and 178-208).

Connexin 45 (SEQ ID NO. 13)Met Ser Trp Ser Phe Leu Thr Arg Leu Leu Glu Glu1               5                   10Ile His Asn His Ser Thr Phe Val Gly Lys Ile Trp        15                  20Leu Thr Val Leu Ile Val Phe Arg Ile Val Leu Thr25                  30                  35Ala Val Gly Gly Glu Ser Ile Tyr Tyr Asp Glu Gln            40                  45Ser Lys Phe Val Cys Asn Thr Glu Gln Pro Gly Cys    50                  55                  60Glu Asn Val Cys Tyr Asp Ala Phe Ala Pro Leu Ser                65                  70His Val Arg Phe Trp Val Phe Gln Ile Ile Leu Val        75                  80Ala Thr Pro Ser Val Met Tyr Leu Gly Tyr Ala Ile85                  90                  95His Lys Ile Ala Lys Met Glu His Gly Glu Ala Asp            100                 105Lys Lys Ala Ala Arg Ser Lys Pro Tyr Ala Met Arg    110                 115                 120Trp Lys Gln His Arg Ala Leu Glu Glu Thr Glu Glu                125                 130Asp Asn Glu Glu Asp Pro Met Met Tyr Pro Glu Met        135                 140Glu Leu Glu Ser Asp Lys Glu Asn Lys Glu Gln Ser145                 150                 155Gln Pro Lys Pro Lys His Asp Gly Arg Arg Arg Ile            160                 165Arg Glu Asp Gly Leu Met Lys Ile Tyr Val Leu Gln    170                 175                 180Leu Leu Ala Arg Thr Val Phe Glu Val Gly Phe Leu                185                 190Ile Gly Gln Tyr Phe Leu Tyr Gly Phe Gln Val His        195                 200Pro Phe Tyr Val Cys Ser Arg Leu Pro Cys Pro His205                 210                 215Lys Ile Asp Cys Phe Ile Ser Arg Pro Thr Glu Lys            220                 225Thr Ile Phe Leu Leu Ile Met Tyr Gly Val Thr Gly    230                 235                 240Leu Cys Leu Leu Leu Asn Ile Trp Glu Met Leu His                245                 250Leu Gly Phe Gly Thr Ile Arg Asp Ser Leu Asn Ser        255                 260Lys Arg Arg Glu Leu Glu Asp Pro Gly Ala Tyr Asn265                 270                 275Tyr Pro Phe Thr Trp Asn Thr Pro Ser Ala Pro Pro            280                 285Gly Tyr Asn Ile Ala Val Lys Pro Asp Gln Ile Gln    290                 295                 300Tyr Thr Glu Leu Ser Asn Ala Lys Ile Ala Tyr Lys                305                 310Gln Asn Lys Ala Asn Thr Ala Gln Glu Gln Gln Tyr        315                 320Gly Ser His Glu Glu Asn Leu Pro Ala Asp Leu Glu325                 330                 335Ala Leu Gln Arg Glu Ile Arg Met Ala Gln Glu Arg            340                 345Leu Asp Leu Ala Val Gln Ala Tyr Ser His Gln Asn    350                 355                 360Asn Pro His Gly Pro Arg Glu Lys Lys Ala Lys Val                365                 370Gly Ser Lys Ala Gly Ser Asn Lys Ser Thr Ala Ser        375                 380Ser Lys Ser Gly Asp Gly Lys Asn Ser Val Trp Ile385                 390                 395 Connexin 43 (SEQ ID NO. 14)Met Gly Asp Trp Ser Ala Leu Gly Lys Leu Leu Asp1               5                   10Lys Val Gln Ala Tyr Ser Thr Ala Gly Gly Lys Val        15                  20Trp Leu Ser Val Leu Phe Ile Phe Arg Ile Leu Leu25                  30                  35Leu Gly Thr Ala Val Glu Ser Ala Trp Gly Asp Glu            40                  45Gln Ser Ala Phe Arg Cys Asn Thr Gln Gln Pro Gly    50                  55                  60Cys Glu Asn Val Cys Tyr Asp Lys Ser Phe Pro Ile                65                  70Ser His Val Arg Phe Trp Val Leu Gln Ile Ile Phe        75                  80Val Ser Val Pro Thr Leu Leu Tyr Leu Ala His Val85                  90                  95Phe Tyr Val Met Arg Lys Glu Glu Lys Leu Asn Lys            100                 105Lys Glu Glu Glu Leu Lys Val Ala Gln Thr Asp Gly    110                 115                 120Val Asn Val Asp Met His Leu Lys Gln Ile Glu Ile                125                 130Lys Lys Phe Lys Tyr Gly Ile Glu Glu His Gly Lys        135                 140Val Lys Met Arg Gly Gly Leu Leu Arg Thr Tyr Ile145                 150                 155Ile Ser Ile Leu Phe Lys Ser Ile Phe Glu Val Ala            160                 165Phe Leu Leu Ile Gln Trp Tyr Ile Tyr Gly Phe Ser    170                 175                 180Leu Ser Ala Val Tyr Thr Cys Lys Arg Asp Pro Cys                185                 190Pro His Gln Val Asp Cys Phe Leu Ser Arg Pro Thr        195                 200Glu Lys Thr Ile Phe Ile Ile Phe Met Leu Val Val205                 210                 215Ser Leu Val Ser Leu Ala Leu Asn Ile Ile Glu Leu            220                 225Phe Tyr Val Phe Phe Lys Gly Val Lys Asp Arg Val    230                 235                 240Lys Gly Lys Ser Asp Pro Tyr His Ala Thr Ser Gly                245                 250Ala Leu Ser Pro Ala Lys Asp Cys Gly Ser Gln Lys        255                 260Tyr Ala Tyr Phe Asn Gly Cys Ser Ser Pro Thr Ala265                 270                 275Pro Leu Ser Pro Met Ser Pro Pro Gly Tyr Lys Leu            280                 285Val Thr Gly Asp Arg Asn Asn Ser Ser Cys Arg Asn    290                 295                 300Tyr Asn Lys Gln Ala Ser Glu Gln Asn Trp Ala Asn                305                 310Tyr Ser Ala Glu Gln Asn Arg Met Gly Gln Ala Gly        315                 320Ser Thr Ile Ser Asn Ser His Ala Gln Pro Phe Asp325                 330                 335Phe Pro Asp Asp Asn Gln Asn Ser Lys Lys Leu Ala            340                 345Ala Gly His Glu Leu Gln Pro Leu Ala Ile Val Asp    350                 355                 360Gln Arg Pro Ser Ser Arg Ala Ser Ser Arg Ala Ser                365                 370Ser Arg Pro Arg Pro Asp Asp Leu Glu Ile         375                 380

The anti-connexin peptides may comprise sequences corresponding to aportion of the connexin extracellular domains with conservative aminoacid substitutions such that peptides are functionally activeanti-connexin agents. Exemplary conservative amino acid substitutionsinclude for example the substitution of a nonpolar amino acid withanother nonpolar amino acid, the substitution of an aromatic amino acidwith another aromatic amino acid, the substitution of an aliphatic aminoacid with another aliphatic amino acid, the substitution of a polaramino acid with another polar amino acid, the substitution of an acidicamino acid with another acidic amino acid, the substitution of a basicamino acid with another basic amino acid, and the substitution of anionizable amino acid with another ionizable amino acid.

Exemplary peptides targeted to connexin 43 are shown below in Table 2.M1, 2, 3 and 4 refer to the 1^(st) to 4^(th) transmembrane regions ofthe connexin 43 protein respectively. E1 and E2 refer to the first andsecond extracellular loops respectively.

TABLE 2 Peptidic Inhibitors of Intercellular Communication (cx43)FEVAFLLIQWI M3 & E2 (SEQ. ID. NO: 15) LLIQWYIGFSL E2 (SEQ. ID. NO: 16)SLSAVYTCKRDPCPHQ E2 (SEQ. ID. NO: 17) VDCFLSRPTEKT E2 (SEQ. ID. NO: 18)SRPTEKTIFII E2 & M4 (SEQ. ID. NO: 19) LGTAVESAWGDEQ M1 & E1(SEQ. ID. NO: 20) QSAFRCNTQQPG E1 (SEQ. ID. NO: 21) QQPGCENVCYDK E1(SEQ. ID. NO: 22) VCYDKSFPISHVR E1 (SEQ. ID. NO: 23)

Table 3 provides additional exemplary connexin peptides used ininhibiting hemichannel or gap junction function. In other embodiments,conservative amino acid changes are made to the peptides or fragmentsthereof.

TABLE 3Additional Peptidic Inhibitors of Intercellular Communication (cx32, cx43)Connexin Location AA's and Sequence Cx32 E1 39-77AAESVWGDEIKSSFICNTLQPGCNSVCYDHFFPISHVR (SEQ. ID. NO: 24) Cx32 E1 41-52ESVWGDEKSSFI (SEQ. ID. NO: 25) Cx32 E1 52-63 ICNTLQPGCNSV(SEQ. ID. NO: 26) Cx32 E1 62-73 SVCYDHFFPISH (SEQ. ID. NO: 27) Cx32E2 64-188 RLVKCEAFPCPNTVDCFVSRPTEKT (SEQ. ID. NO: 28) Cx32 E2 166-177VKCEAFPCPNTV (SEQ. ID. NO: 29) Cx32 E2 177-188 VDCFVSRPTEKT(SEQ. ID. NO: 30) Cx32 E1 63-75 VCYDHFFPISHVR (SEQ. ID. NO: 31) Cx32E1 45-59 VWGDEKSSFICNTLQPGY (SEQ. ID. NO: 32) Cx32 E1 46-59DEKSSFICNTLQPGY (SEQ. ID  NO: 33) Cx32 E2 182-192 SRPTEKTVFTV(SEQ. ID. NO: 34) Cx32/Cx43 E2 182-188/ SRPTEKT (SEQ. ID. NO: 35)201-207 Cx32 E1 52-63 ICNTLQPGCNSV (SEQ. ID. NO: 36) Cx40 E2 177-192FLDTLHVCRRSPCPHP (SEQ. ID. NO: 37) Cx43 E2 188-205 KRDPCHQVDCFLSRPTEK(SEQ. ID. NO: 38)

Table 4 provides the extracellular loops for connexin family memberswhich are used to develop peptide inhibitors for use as describedherein. The peptides and provided in Table 4, and fragments thereof, areused as peptide inhibitors in certain non-limiting embodiments. In othernon-limiting embodiments, peptides comprising from about 8 to about 15,or from about 11 to about 13 amino contiguous amino acids of thepeptides in this Table 4 are peptide inhibitors. Conservative amino acidchanges may be made to the peptides or fragments thereof

TABLE 4 Extracellular loops for various connexin family members E1huCx26 KEVWGDEQADFVCNTLQPGCKNVCYDHYFPISHIR (SEQ. ID. NO: 39) huCx30QEVWGDEQEDFVCNTLQPGCKNVCYDHFFPVSHIR (SEQ. ID. NO: 40) huCx30.3EEVWDDEQKDFVCNTKQPGCPNVCYDEFFPVSHVR (SEQ. ID. NO: 41) huCx31ERVWGDEQKDFDCNTKQPGCTNVCYDNYFPISNIR (SEQ. ID. NO: 42) huCx31.1ERVWSDDHKDFDCNTRQPGCSNVCFDEFFPVSHVR (SEQ. ID. NO: 43) huCx32ESVWGDEKSSFICNTLQPGCNSVCYDQFFPISHVR (SEQ. ID. NO: 44) huCx36ESVWGDEQSDFECNTAQPGCTNVCYDQAFPISHIR (SEQ. ID. NO: 45) huCx37ESVWGDEQSDFECNTAQPGCTNVCYDQAFPISHIR (SEQ. ID. NO: 46) huCx40.1RPVYQDEQERFVCNTLQPGCANVCYDVFSPVSHLR (SEQ. ID. NO: 47) huCx43ESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVR (SEQ. ID. NO: 48) huCx46EDVWGDEQSDFTCNTQQPGCBNVCYBRAFPISHIR (SEQ. ID. NO: 49) huCx46.6EAIYSDEQAKFTCNTRQPGCDNVCYDAFAPLSHVR (SEQ. ID. NO: 50) huCx40ESSWGDEQADFRCDTIQPGCQNVCTDQAFPISHIR (SEQ. ID. NO: 51) huCx45GESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVR (SEQ. ID. NO: 52) E2 huCx26MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKT (SEQ. ID. NO: 53) huCx30MYVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKT (SEQ. ID. NO: 54) huCx30.3LYIFHRLYKDYDMPRVVACSVEPCPHTVDCYISRPTEKK (SEQ. ID. NO: 55) huCx31LYLLHTLWHGFNMPRLVQCANVAPCPNIVDCYIARPTEKK (SEQ. ID. NO: 56) huCx31.1LYVFHSFYPKYILPPVVKCHADPCPNIVDCFISKPSEKN (SEQ. ID. NO: 57) huCx32MYVFYLLYPGYAMVRLVKCDVYPCPNTVDCFVSRPTEKT SEQ. ID. NO: 58) huCx36LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKT (SEQ. ID. NO: 59) huCx37LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKT (SEQ. ID. NO: 60) huCx40.1GALHYFLFGFLAPKKFPCTRPPCTGVVDCYVSRPTSKS (SEQ. ID. NO: 61) huCx43LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKT (SEQ. ID. NO: 62) huCx46IAGQYFLYGFELKPLYRCDRWPCPNTVDCFISRPTEKT (SEQ. ID. NO: 63) huCx46.6LVGQYLLYGFEVRPFFPCSRQPCPHVVDCFVSRPTEKT (SEQ. ID. NO: 64) huCx40IVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKN (SEQ. ID. NO: 65) huCx45LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKT (SEQ. ID. NO: 66)

Table 5 provides the extracellular domain for connexin family memberswhich may be used to develop peptide anti-connexin agents. The peptidesand provided in Table 5, and fragments thereof, may also be used aspeptide anti-connexin agents. Such peptides may comprise from about 8 toabout 15, or from about 11 to about 13 amino contiguous amino acids ofthe peptide sequence in this Table 5. Conservative amino acid changesmay be made to the peptides or fragments thereof.

TABLE 5 Extracellular domains Peptide VDCFLSRPTEKT (SEQ. ID. NO: 18)Peptide SRPTEKTIFII (SEQ. ID. NO: 19) huCx43LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFII (SEQ. ID. NO: 67) huCx26MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKTVFTV (SEQ. ID. NO: 68) huCx30YVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKTVFTI (SEQ. ID. NO: 69) huCx30.3LYIFHRLYKDYDMPRVVACSVEPCPHTVDCYISRPTEKKVFTY (SEQ. ID. NO: 70) huCx31LYLLHTLWHGFNMPRLVQCANVAPCPNIVDCYIARPTEKKTY (SEQ. ID. NO: 71) huCx31.1LYVFHSFYPKYILPPVVKCHADPCPNIVDCFISKPSEKNIFTL (SEQ. ID. NO: 72) huCx32MYVFYLLYPGYAMVRLVKCDVYPCPNTVDCFVSRPTEKTVFTV (SEQ. ID. NO: 73) huCx36LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKTIFII (SEQ. ID. NO: 74) huCx37LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKTIFII (SEQ. ID. NO: 75) huCx40.1GALHYFLFGFLAPKKFPCTRPPCTGVVDCYVSRPTEKSLLML (SEQ. ID. NO: 76) huCx46IAGQYFLYGFELKPLYRCDRWPCPNTVDCFISRPTEKTIFII (SEQ. ID. NO: 77) huCx46.6LVGQYLLYGFEVRPFFPCSRQPCPHVVDCFVSRPTEKTVFLL (SEQ. ID. NO: 78) huCx40IVGQYFIYGIFLTTLHVCRRSPCPHPVNCYSRPTEKNVFIV (SEQ. ID. NO: 79) huCx45LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL (SEQ. ID. NO: 80)

Table 6 provides peptides inhibitors of connexin 40 shown with referenceto the extracellular loops (E1 and E2) of connexin 40. The bold aminoacids are directed to the transmembrane regions of connexin 40.

TABLE 6 Cx40 peptide inhibitors E2LGTAAESSWGDEQADFRCDTIQPGCQNVCTDQAFPISHIRFWVLQ (SEQ. ID. NO: 81)LGTAAESSWGDEQA (SEQ. ID. NO: 82)           DEQADFRCDTIQP(SEQ. ID. NO: 83)                    TIQPGCQNVCTDQ (SEQ. ID. NO: 84)                           VCTDQAFPISHIR (SEQ. ID. NO: 85)                                AFPISHIRFWVLQ (SEQ. ID. NO: 86) E2MEVGFIVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKNVFIV (SEQ. ID. NO: 87)MEVGFIVGQYF (SEQ. ID. NO: 88)      IVGQYFIYGIFL (SEQ. ID. NO: 89)             GIFLTTLHVCRRSP (SEQ. ID. NO: 90)                       RRSPCPHPVNCY (SEQ. ID. NO: 91)                               VNCYVSRPTEKN (SEQ. ID. NO: 92)                                    SRPTEKNVFIV (SEQ. ID. NO: 93)

Table 7 provides peptides inhibitors of connexin 45 shown with referenceto the extracellular loops (E1 and E2) of connexin 45. The bold aminoacids are directed to the transmembrane regions of connexin 45

TABLE 7 Cx45 peptide inhibitors E1LTAVGGESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVRFWVFQ (SEQ. ID. NO: 94)LTAVGGEISIYYDEQS (SEQ. ID. NO: 95)      DEQSKFVCNTEQP (SEQ. ID. NO: 96)       TEQPGCENVCYDA (SEQ. ID. NO: 97)           VCYDAFAPLSHVR(SEQ. ID. NO: 98)             APLSHVRFWVFQ (SEQ. ID. NO: 99) E2FEVGFLIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL (SEQ. ID. NO: 100)FEVGFLIGQYF (SEQ. ID. NO: 101)   LIGQYFLYGFQV (SEQ. ID. NO: 102)     GFQVHPFYVCSRLP (SEQ. ID. NO: 103)          SRLPCHPKIDCF(SEQ. ID. NO: 104)             IDCFISRPTEKT (SEQ. ID. NO: 105)              SRPTEKTIFLL (SEQ. ID. NO: 106)

In certain embodiments, it is preferred that certain peptide inhibitorsblock hemichannels without disrupting existing gap junctions. While notwishing to be bound to any particular theory or mechanism, it is alsobelieved that certain peptidomimetics (e.g. VCYDKSFPISHVR, (SEQ.ID.NO:23) block hemichannels without causing uncoupling of gap junctions (SeeLeybeart et al., Cell Commun. Adhes. 10: 251-257 (2003)), or do so inlower dose amounts. The peptide SRPTEKTIFII (SEQ.ID.NO: 19) may also beused, for example to block hemichannels without uncoupling of gapjunctions. The peptide SRGGEKNVFIV (SEQ.ID.NO: 107) may be used that asa control sequence (DeVriese et al., Kidney Internat. 61: 177-185(2002)). Examples of peptide inhibitors for connexin 45 YVCSRLPCHP(SEQ.ID.NO:108), QVHPFYVCSRL (SEQ.ID.NO:109), FEVGFLIGQYFLY(SEQ.ID.NO:110), GQYFLYGFQVHP (SEQ.ID.NO:111), GFQVHPFYVCSR(SEQ.ID.NO:112), AVGGESIYYDEQ (SEQ.ID.NO), YDEQSKFVCNTE (SEQ.ID.NO:114),NTEQPGCENVCY (SEQ.ID.NO:115), CYDAFAPLSHVR (SEQ.ID.NO:116), FAPLSHVRFWVF(SEQ.ID.NO:117) and LIGQY (SEQ.ID.NO:118), QVHPF (SEQ.ID.NO:119), YVCSR(SEQ.ID.NO:120), SRLPC (SEQ.ID.NO:121), LPCHP (SEQ.ID.NO:122) and GESIY(SEQ.ID.NO:123), YDEQSK (SEQ.ID.NO:124), SKFVCN (SEQ.ID.NO:125),TEQPGCEN (SEQ.ID.NO:126), VCYDAFAP (SEQ.ID.NO:127), LSHVRFWVFQ(SEQ.ID.NO:128) The peptides may only be 3 amino acids in length,including SRL, PCH, LCP, CHP, IYY, SKF, QPC, VCY, APL, HVR, or longer,for example: LIQYFLYGFQVHPF (SEQ.ID.NO:129), VHPFYCSRLPCHP(SEQ.ID.NO:130), VGGESIYYDEQSKFVCNTEQPG (SEQ.ID.NO:131),TEQPGCENVCYDAFAPLSHVRF (SEQ.ID.NO:132), AFAPLSHVRFWVFQ (SEQ.ID.NO: 133).

TABLE 8 Table 8A Human Connexin 43 from GenBank Accession No. M65188(SEQ. ID. NO: 134) 1ggcttttagc gtgaggaaag taccaaacag cagcggagtt ttaaacttta aatagacagg 61tctgagtgcc tgaacttgcc ttttcatttt acttcatcct ccaaggagtt caatcacttg 121gcgtgacttc actactttta agcaaaagag tggtgcccag gcaacatggg tgactggagc 181gccttaggca aactccttga caaggttcaa gcctactcaa ctgctggagg gaaggtgtgg 241ctgtcagtac ttttcatttt ccgaatcctg ctgctgggga cagcggttga gtcagcctgg 301ggagatgagc agtctgcctt tcgttgtaac actcagcaac ctggttgtga aaatgtctgc 361tatgacaagt ctttcccaat ctctcatgtg cgcttctggg tcctgcagat catatttgtg 421tctgtaccca cactcttgta cctggctcat gtgttctatg tgatgcgaaa ggaagagaaa 481ctgaacaaga aagaggaaga actcaaggtt gcccaaactg atggtgtcaa tgtggacatg 541cacttgaagc agattgagat aaagaagttc aagtacggta ttgaagagca tggtaaggtg 601aaaatgcgag gggggttgct gcgaacctac atcatcagta tcctcttcaa gtctatcttt 661gaggtggcct tcttgctgat ccagtggtac atctatggat tcagcttgag tgctgtttac 721acttgcaaaa gagatccctg cccacatcag gtggactgtt tcctctctcg ccccacggag 781aaaaccatct tcatcatctt catgctggtg gtgtccttgg tgtccctggc cttgaatatc 841attgaactct tctatgtttt cttcaagggc gttaaggatc gggttaaggg aaagagcgac 901ccttaccatg cgaccagtgg tgcgctgagc cctgccaaag actgtgggtc tcaaaaatat 961gcttatttca atggctgctc ctcaccaacc gctcccctct cgcctatgtc tcctcctggg 1021tacaagctgg ttactggcga cagaaacaat tcttcttgcc gcaattacaa caagcaagca 1081agtgagcaaa actgggctaa ttacagtgca gaacaaaatc gaatggggca ggcgggaagc 1141accatctcta actcccatgc acagcctttt gatttccccg atgataacca gaattctaaa 1201aaactagctg ctggacatga attacagcca ctagccattg tggaccagcg accttcaagc 1261agagccagca gtcgtgccag cagcagacct cggcctgatg acctggagat ctag

TABLE 8B Human Connexin 43 (SEQ. ID. NO: 135) 1atgggtgactggagcgcctt aggcaaactc cttgacaagg ttcaagtcta ctcaactgct 61ggagggaaggtgtggctgtc agtacttttc attttccgaatcctgctgct ggggacagcg 121gttgagtcagcctggggaga tgagcagtct gcctttcgtt gtaacactca gcaacctggt 181tgtgaaaatg tctgctatga caagtctttcccaatctctc atgtgcgctt ctgggtcctg 241cagatcatat ttgtgtctgt acccacactcttgtacctgg ctcatgtgttctatgtgatg 301cgaaaggaag agaaactgaa caagaaagag gaagaactca aggttgccca aactgatggt 361gtcaatgtgg acatgcactt gaagcagatt gagataaagaagttcaagta cggtattgaa 421gagcatggta aggtgaaaat gcgagggggg ttgctgcgaa cctacatcat cagtatcctc 481ttcaagtcta tctttgaggt ggccttcttg ctgatccagt ggtacatcta tggattcagc 541ttgagtgctg tttacacttg caaaagagat ccctgcccac atcaggtgga ctgtttcctc 601tctcgcccca cggagaaaac catcttcatc atcttcatgc tggtggtgtc cttggtgtcc 661ctggccttga atatcattga actcttctat gttttcttca agggcgttaa ggatcgggtt 721aagggaaaga gcgaccctta ccatgcgacc agtggtgcgc tgagccctgc caaagactgt 781gggtctcaaa aatatgctta tttcaatggc tgctcctcac caaccgctcc cctctcgcct 841atgtctcctc ctgggtacaa gctggttact ggcgacagaa acaattcttc ttgccgcaat 901tacaacaagc aagcaagtga gcaaaactgg gctaattaca gtgcagaaca aaatcgaatg 961gggcaggcgg gaagcaccat ctctaactcc catgcacagccttttgattt ccccgatgat 1021aaccagaatt ctaaaaaactagctgctgga catgaattac agccactagc cattgtggac 1081cagcgacctt caagcagagc cagcagtcgtgccagcagca gacctcggcctgatgacctg 1141gagatctag

Therapeutic Agents

Therapeutic agents include pharmaceutically acceptable agents useful inthe treatment of wounds or the promotion of wound-healing, whethercurrently existing and known or later developed. Therapeutic agentsinclude, for example, anti-infectives, anesthetics, analgesics,antibiotics, narcotics, and steroidal and non-steroidalanti-inflammatory agents. Preferred therapeutic agents include topicalsteroid anti-inflammatory agents, antimicrobial agents, local andtopical anesthetics, and topical opioids.

Therapeutic agents may include, for example, anti-thrombogenic agents,anticoagulants, antiplatelet agents, thrombolytics, antiproliferatives,anti-inflammatory agents, statins, α-adrenergic receptor antagonists,β₁-selective adrenergic antagonists, ACE inhibitors, calcium channelblockers, angiotensin II receptor antagonists, vasodilators,anti-proliferative/antimitotic agents, immunosuppressive agent, agentsthat inhibit hyperplasia and in particular restenosis, smooth musclecell inhibitors, antibiotics, cytokines, growth factors, growth factorinhibitors, cell adhesion inhibitors, cell adhesion promoters and drugsthat may enhance the formation of healthy tissue, including endothelialand epithelial cell regeneration, etc.

Anti-thrombogenic agents, may include, for example, heparin, warfarin,hirudin and its analogs, aspirin, indomethacin, dipyridamole,prostacyclin, prostaglandin E, sulfinpyrazone, abciximab, eptifabatide,phenothiazines (such as chlorpromazine or trifluperazine) RGD(arginine-glycine-aspartic acid) peptide or RGD peptide mimetics, agentsthat block platelet glycoprotein IIb-IIIa receptors (such as C-7E3),ticlopidine or the thienopyridine known as clopidogrel. Statins, mayinclude, for example, simvastatin, atorvastatin, lovastatin,pravastatin, and fluvastatin. α-adrenergic receptor antagonists mayinclude, for example, prazosin, terazosin, doxazosin, ketanserin,indoramin, urapidil, clonideine, guanabenz, guanfacine, guanadrel,reserpine, and metyrosine. β₁-selective adrenergic antagonists mayinclude, for example, metoprolol, atenolol, esmolol, acebutolol,bopindolol, carteolol, oxprenolol, penbutolol, medroxalol, bucindolol,levobunolol, metipranolol, bisoprolol, nebivolol, betaxolol, celiprolol,sotalol, propafenone, propranolol, timolol maleate, and nadolol. ACEinhibitors may include, for example, captopriol, fentiapril, pivalopril,zofenopril, alacepril, enalapril, enalaprilat, enalaprilo, lisinopril,benazepril, quinapril, moexipril. Calcium channel blockers may include,for example, nisoldipine, verapamil, diltiazem, nifedipine, nimodipine,felodipine, nicardipine, isradipine, amlodipine, and bepridil.Angiotensin H receptor antagonists may include, for example, losartan,candesartan, irbesartan, valsartan, telmisartan, eprosartan, andolmesartan medoxomil. Vasodilators may include, for example,hydralazine, minoxidil, sodium nitroprusside, diazoxide, bosentan,eporprostenol, treprostinil, and iloprost. Anti-inflammatory agents mayinclude, for example, steroids (including, for example, cortisol,cortisone, fludrocortisone, prednisone, prednisolone,6-α-methylprednisolone, triamcinolone, betamethasone, corticosterone,budesonide, estrogen, sulfasalazine, mesalamine and dexamethasone),non-steroidal agents (including, for example, salicylic acid derivativesi.e. aspirin; para-aminophenol derivatives e.g. acetaminophen; indoleand indene acetic acids, e.g. indomethacin, sulindac, and etodalac;heteroaryl acetic acids e.g. tolmetin, diclofenac, and ketorolac;arylpropionic acids e.g. ibuprofen and derivatives; anthranilic acidse.g. mefenamic acid, and meclofenamic acid; enolic acids e.g. piroxicam,tenoxicam, phenylbutazone, and oxyphenthatrazone); and nabumetone.Imunnosuppresant agents may include, for example, sirolimus,cyclosporine, tacrolimus, azathioprine, and mycophenolate mofetil.Anti-proliferative/antimitotic agents may also be used as therapeuticagents, including, for example, such as vinca alkaloids (e.g.vinblastine, vincristine, and vinorelbine), paclitaxel,epidipodophyllotoxins (e.g. etoposide, teniposide), antibiotics(dactinomycin (actinomycin D), daunorubicin, doxorubicin andidarubicin), anthracyclines, mitoxantrone, bleomycins, plicamycin(mithramycin) and mitomycin. Antiplatelet agents including, for example,acetylsalicylic acid, dipyridamole, clopidogrel, ticlopidine, abciximab,eptifbatide, tirofiban, reversable COX-1 inhibitors, BPIIIb/IIIablockers, TP antagonists, and P2Y12 antagonists.

Anti-inflammatory agents may include, for example, steroids (including,for example, cortisol, cortisone, fludrocortisone, prednisone,prednisolone, 6-α-methylprednisolone, triamcinolone, betamethasone,corticosterone, budesonide, estrogen, sulfasalazine, mesalamine anddexamethasone), non-steroidal agents (including, for example, salicylicacid derivatives i.e. aspirin; para-aminophenol derivatives e.gacetaminophen; indole and indene acetic acids, e.g. indomethacin,sulindac, and etodalac; heteroaryl acetic acids e.g. tolmetin,diclofenac, and ketorolac; arylpropionic acids e.g ibuprofen andderivatives; anthranilic acids e.g mefenamic acid, and meclofenamicacid; enolic acids e.g piroxicam, tenoxicam, phenylbutazone, andoxyphenthatrazone); and nabumetone.

Imunnosuppresant agents may include, for example, sirolimus,cyclosporine, tacrolimus, azathioprine, and mycophenolate mofetil.

Appropriate dose levels for such therapeutic agents are known to thosein the pharmaceutical arts. Such therapeutic agents may be prepared andformulated for inclusion in the medical devices of the present inventionusing techinques known to those in the art.

In certain embodiments, one, two three, four, five or six therapeuticagents may be used in combination.

Agents Useful for Wound Healing

As used herein, agents useful for wound healing include stimulators,enhancers or positive mediators of the wound healing cascade which 1)promote or accelerate the natural wound healing process or 2) reduceeffects associated with improper or delayed wound healing, which effectsinclude, for example, adverse inflammation, epithelialization,angiogenesis and matrix deposition, and scarring and fibrosis.

Positive mediators, enhancers and stimulators include for example, anagent which may stimulate, enhance, facilitate, or accelerate (i.e.,agonize) the quantity, quality or efficacy of wound healing or theactive wound healing process, or a wound healing-associated growthfactor or cytokine at a wound site, or the activation of a woundhealing-associated growth factor or cytokine receptor. Such agents mayinclude a wound healing-associated growth factor or cytokine or apartially modified form of a wound healing-associated growth factor orcytokine, for example. A partially modified form of woundhealing-associated growth factor or cytokine may, for example, have alonger half-life than the natural wound healing-associated growth factoror cytokine. Alternatively, it may be an inhibitor of woundhealing-associated growth factor or cytokine metabolism.

Partial modification of such an agent may be by way of addition,deletion or substitution of amino acid residues. A substitution may forexample be a conserved substitution. Hence a partially modified moleculemay be a homologue of the molecule from which it was derived. It mayhave at least about 40%, for example about 50, 60, 70, 80, 90 or 95%,homology with the molecule from which it is derived.

As used herein, agents useful for wound healing may include for example,wound-healing-promoting or scar-reducing agents for wound treatmentmodalities now known in the art or later-developed; exemplary factors,agents or modalities including natural or synthetic growth factors,cytokines, or modulators thereof to promote wound healing, wound healingpromoting bioengineered matrix, dressings bandages, and the like.Suitable examples may include, but not limited to 1) topical or dressingand related therapies and debriding agents (such as, for example,Santyl® collagenase) and Iodosorb® (cadexomer iodine); 2) antimicrobialagents, including systemic or topical creams or gels, including, forexample, silver-containing agents such as SAGs (silver antimicrobialgels), (CollaGUARD™), Innocoll, Inc) (purified type-I collagen proteinbased dressing), CollaGUARD Ag (a collagen-based bioactive dressingimpregnated with silver for infected wounds or wounds at risk ofinfection), DermaSIL™ (a collagen-synthetic foam composite dressing fordeep and heavily exuding wounds); 3) cell therapy or bioengineered skin,skin substitutes, and skin equivalents, including, for example,Dermograft (3-dimensional matrix cultivation of human fibroblasts thatsecrete cytokines and growth factors), Apligraf® (human keratinocytesand fibroblasts), Graftskin® (bilayer of epidermal cells and fibroblaststhat is histologically similar to normal skin and produces growthfactors similar to those produced by normal skin), TransCyte (a HumanFibroblast Derived Temporary Skin Substitute) and Oasis® (an activebiomaterial that comprises both growth factors and extracellular matrixcomponents such as collagen, proteoglycans, and glycosaminoglycans); 4)cytokines, growth factors or hormones (both natural and synthetic)introduced to the wound to promote wound healing, including, forexample, NGF, NT3, BDGF, integrins, plasmin, semaphoring, blood-derivedgrowth factor, keratinocyte growth factor, tissue growth factor,TGF-alpha, TGF-beta, PDGF (one or more of the three subtypes may beused: AA, AB, and B), PDGF-BB, TGF-beta 3, factors that modulate therelative levels of TGFβ3, TGFβ1, and TGFβ2 (e.g., Mannose-6-phosphate),sex steroids, including for example, estrogen, estradiol, or anoestrogen receptor agonist selected from the group consisting ofethinyloestradiol, dienoestrol, mestranol, oestradiol, oestriol, aconjugated oestrogen, piperazine oestrone sulphate, stilboestrol,fosfesterol tetrasodium, polyestradiol phosphate, tibolone, aphytoestrogen, 17-beta-estradiol; thymic hormones such asThymosin-beta-4, EGF, HB-EGF, fibroblast growth factors (e.g., FGF1,FGF2, FGF7), keratinocyte growth factor, TNF, interleukins family ofinflammatory response modulators such as, for example, IL-10, IL-1,IL-2, IL-6, IL-8, and IL-10 and modulators thereof; INFs (INF-alpha,-beta, and -delta); stimulators of activin or inhibin, and inhibitors ofinterferon gamma prostaglandin E2 (PGE2) and of mediators of theadenosine 3′,5′-cyclic monophosphate (cAMP) pathway; adenosine A1agonist, adenosine A2 agonist or 5) other agents useful for woundhealing, including, for example, both natural or synthetic homologues,agonist and antagonist of VEGF, VEGFA, IGF; IGF-1, proinflammatorycytokines, GM-CSF, and leptins and 6) IGF-1 and KGF cDNA, autologousplatelet gel, hypochlorous acid (Sterilox® lipoic acid, nitric oxidesynthase3, matrix metalloproteinase 9 (MMP-9), CCT-ETA, alphavbeta6integrin, growth factor-primed fibroblasts and Decorin, silvercontaining wound dressings, Xenaderm™, papain wound debriding agents,lactoferrin, substance P, collagen, and silver-ORC, placental alkalinephosphatase or placental growth factor, modulators of hedgehogsignaling, modulators of cholesterol synthesis pathway, and APC(Activated Protein C), keratinocyte growth factor, TNF, Thromboxane A2,NGF, BMP bone morphogenetic protein, CTGF (connective tissue growthfactor), wound healing chemokines, decorin, modulators of lactateinduced neovascularization, cod liver oil, placental alkalinephosphatase or placental growth factor, and thymosin beta 4. In certainembodiments, one, two three, four, five or six agents useful for woundhealing may be used in combination.

It is to be understood that the agents useful for wound healing(including for example, growth factors and cytokines) above encompassall naturally occurring polymorphs (for example, polymorphs of thegrowth factors or cytokines). Also, functional fragments, chimericproteins comprising one of said agents useful for wound healing or afunctional fragment thereof, homologues obtained by analogoussubstitution of one or more amino acids of the wound healing agent, andspecies homologues are encompassed. It is contemplated that one or moreagents useful for wound healing may be a product of recombinant DNAtechnology, and one or more agents useful for wound healing may be aproduct of transgenic technology. For example, platelet derived growthfactor may be provided in the form of a recombinant PDGF or a genetherapy vector comprising a coding sequence for PDGF.

A fragment or partially modified form thereof refers to a fragment orpartially modified form of the wound healing agent which retains thebiological or wound healing functionality of the factor, although it mayof course have additional functionality. Partial modification may, forexample, be by way of addition, deletion or substitution of amino acidresidues. For example, a substitution may be a conserved substitution.Hence the partially modified molecules may be homologues of the woundhealing agent. They may, for example, have at least about 40% homologywith said factor. They may for example have at least about 50, 60, 70,80, 90 or 95% homology with said factor. For example, in certainembodiments, IL-10 or a fragment or a partially modified form thereofmay be administered at a concentration of between about 1 μM and about10 μM. It may be administered at a concentration of between about 2.5 μMand about 5 μM. In certain other embodiments, IL-10 or a fragment or apartially modified form thereof may be administered immediately prior towound healing, but may be effective if administered within about 7 daysof wounding. It could be administered on at least two occasions.

Gap Junction Modifying Agents

Gap junction modifying agents, include agents that close or block gapjunctions or otherwise prevent or decrease cell to cell communicationvia gap junctions.

As used herein, “gap junction modifying agent” may broadly include thoseagents or compounds that prevent, decrease or modulate, in whole or inpart, the activity, function, or formation of a hemichannel or a gapjunction.

In other embodiments, a gap junction modifying agent prevents ordecreases, in whole or in part, the formation or activity of ahemichannel or a gap junction.

In certain embodiments, a gap junction modifying agent induces closure,in whole or in part, of a hemichannel or a gap junction. In otherembodiments, a gap junction modifying agent blocks, in whole or in part,a hemichannel or a gap junction. In certain embodiments, a gap junctionmodifying agent decreases or prevents, in whole or in part, the openingof a hemichannel or gap junction.

In certain embodiments, said blocking or closure of a gap junction orhemichannel by a gap junction modifying agent can reduce or inhibitextracellular hemichannel communication by preventing or decreasing theflow of small molecules through an open channel to and from anextracellular or periplamic space.

Gap junction modifying agents used for closing gap junctions (e.g.phosphorylating connexin 43 tyrosine residue) have been reported in U.S.Pat. No. 7,153,822 to Jensen et al., U.S. Pat. No. 7,250,397, andassorted patent publications. Exemplary gap junction modifying agents,also include peptides and peptidomimetics are reported in Green et al.,WO2006134494. See also Gourdie et al., see WO2006069181, and Tudor etal., see WO2003032964.

As used herein, “gap junction phosphorylating agent” may include thoseagents or compounds capable of inducing phosphorylation on connexinamino acid residues in order to induce gap junction or hemichannelclosure. Exemplary sites of phosphorylation include one or more of atyrosine, serine or threonine residues on the connexin protein. Incertain embodiments, modulation of phosphorylation may occur on one ormore residues on one or more connexin proteins. Exemplary gap junctionphosphorylating agents are well known in the art and may include, forexample, c-Src tyrosine kinase or other G protein-coupled receptoragonists. See Giepmans B, J. Biol. Chem., Vol. 276, Issue 11, 8544-8549,Mar. 16, 2001. In one embodiment, modulation of phosphorylation on oneor more of these residues impacts hemichannel function, particularly byclosing the hemichannel. In another embodiment, modulation ofphosphorylation on one or more of these residues impacts gap junctionfunction, particularly by closing the gap junction. Gap junctionphosphorylating agents that target the closure of connexin 43 gapjunctions and hemichannels are preferred. Still other anti-connexinagents include connexin carboxy-terminal polypeptides. See Gourdie etal., WO2006/069181.

In certain another aspect, gap junction modifying agent may include, forexample, aliphatic alcohols; octanol; heptanol; anesthetics (e.g.halothane), ethrane, fluothane, propofol and thiopental; anandamide;arylaminobenzoate (FFA: flufenamic acid and similar derivatives that arelipophilic); carbenoxolone; Chalcone: (2′,5′-dihydroxychalcone); CHFs(Chlorohydroxyfuranones); CMCF(3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone); dexamethasone;doxorubicin (and other anthraquinone derivatives); eicosanoidthromboxane A(2) (TXA(2)) mimetics; NO (nitric oxide); Fatty acids (e.g.arachidonic acid, oleic acid and lipoxygenase metabolites; Fenamates(flufenamic (FFA), niflumic (NFA) and meclofenamic acids (MFA));Genistein; glycyrrhetinic acid (GA):18a-glycyrrhetinic acid and18-beta-glycyrrhetinic acid, and derivatives thereof; lindane;lysophosphatidic acid; mefloquine; menadione;2-Methyl-1,4-naphthoquinone, vitamin K(3); nafenopin; okadaic acid;oleamide; oleic acid; PH, gating by intracellular acidification; e.g.acidifying agents; polyunsaturated fatty acids; fatty acid GJICinhibitors (e.g. oleic and arachidonic acids); quinidine; quinine; alltrans-retinoic acid; and tamoxifen.

Medical Devices

The term “medical device” refers to an instrument, apparatus, implement,machine, contrivance, implant, or other similar or related article,including a component part or accessory which is intended for use in thediagnosis of disease or other conditions or in the cure, mitigation,treatment or prevention of a disease, disorder or condition in humans orin other animals (particularly in mammals); is intended to affect thestructure or any function of the body of a human or other animal; or isrecognized in the official National Formulary or the United StatesPharmacopoeia or any supplement to them. In particular, “medical device”includes a manufactured product which is used to cope with a disease,disorder or condition (particularly human diseases, disorders orconditions), such as by preventing, diagnosing, treating, alleviatingand or monitoring disease, disorder or condition; care for injuries(particularly human injuries), such as by diagnosing, treating,alleviating, monitoring or compensating for injuries; meet anatomicalneeds (particularly human anatomical needs), such as by investigating,replacing, modifying or supporting anatomical structures; maintainphysiological functions (particularly human physiological functions),such as by investigating, replacing, modifying or supportingphysiological functions; supporting or sustaining life (particularlyhuman life); and controlling conception (particularly human conception).

The term “implantable medical device” refers to a medical device that ispartly or totally inserted into the subject's body (such as the body ofa human or other mammal) or a natural orifice thereof and is expected tostay there for an extended period of time (for example about 2, 5 7, 10,14, or 30 days or more). Typically, surgical or medical procedures areused to insert or apply implantable medical devices, and surgical ormedical procedures are used to remove them.

The anti-connexin agents, compositions, and methods provided herein canbe used in a variety of procedures that utilize of implants, medical andsurgical devices, and the like. In one aspect, implants, surgicaldevices or stents, are coated with or otherwise constructed to containand/or release any of the anti-connexin agents provided herein. Medicaldevices comprising an anti-connexin agent, may include, for example,stents, balloons, prosthetic heart valves, annuloplasty rings,ventricular assist devices, including left ventricular assist devices,right ventricular assist devices, and biventricular assist devices,grafts, shunts, sewing rings (including those having silicone orpolyurethane inserts), polyester fabric encasements, medical leads,orthopedic plates, bone pins, bone substitutes, anchors, joints, screws,ophthalmic implants (including, for example, orbital implants, lensimplants, corneal implants (including intrasomal corneal ring segments(INTACS)), and microchips), catheters, cannulae, pulse generators,cardiac defibrillators, arteriovenous shunts, pacemakers, sutures,suture anchors, staples, anastomosis devices, vertebral disks,hemostatic barriers, clamps, clips, vascular implants, tissue adhesivesand sealants, tissue scaffolds, intraluminal devices, vascular supportsas well as any devices used in connection with general orthopedic, hipreplacement, CNS, ocular, gastrointestinal, and endoscopy procedures.

Other examples include cardiovascular devices (e.g., implantable venouscatheters, venous ports, tunneled venous catheters, chronic infusionlines or ports, including hepatic artery infusion catheters, pacemakerwires, implantable defibrillators); neurologic/neurosurgical devices(e.g., ventricular peritoneal shunts, ventricular atrial shunts, nervestimulator devices, dural patches and implants to prevent epiduralfibrosis post-laminectomy, devices for continuous subarachnoidinfusions); gastrointestinal devices (e.g., chronic indwellingcatheters, feeding tubes, and shunts), splints for faileddacrocystalrhinostomy, implants for diabetic retinopathy; otolaryngologydevices (e.g., ossicular implants, Eustachian tube splints or stents forglue ear or chronic otitis as an alternative to transtempanic drains);plastic surgery implants (e.g., prevention of fibrous contracture inresponse to gel- or saline-containing breast implants in the subpectoralor subglandular approaches or post-mastectomy, or chin implants), andorthopedic implants (e.g., cemented orthopedic prostheses).

Examples of stents include intravascular and intraductal stents, and seealso, for example, Pepine et al., “Coronary Artery Stents, JACC Vol. 28,No. 3, September 1996:782-94; D. Stoeckel, “A survey of stent designs,”Min Invas Ther & Allied Technol 2002: 11(4) 137-147. Stents may alsoinclude, for example, balloon-expandable stents and self-expandingstents. Balloon-expandable stents include those of the sort availablefrom a number of commercial suppliers, including Cordis Johnson &Johnson Interventional Systems, Medi-Tech, Cook, ACS, and Metronic.Self-expanding stents are typically composed, for example, from a shapememory alloy and are available from suppliers, such as Instent. In thecase of stents, a balloon-expandable stent is typically composed of astainless steel framework or, in the case of self-expanding stents, fromnickel/titanium alloy. While typically stents are made of a metallicmaterial for strength purposes, polymeric or plastic materials may alsobe utilized in the stent construction. While it is preferred that thestent be coated in an expanded position, coating the stent in anunexpanded position is also contemplated. Exemplary coated balloonsinclude, for example, coated balloons and coated balloon catheters,including inflatable and self inflatable coated balloons and ballooncatheters. The inflatable coated balloon may be a non-dispensableballoon, for example, typically composed of polyethyleneterephthalate,or it may be an elastic balloon, for example, typically being composedof latex or silicone rubber.

Preparation of Devices

Implants and other surgical and medical devices may be coated with (orotherwise adapted to release) agents of the invention (e.g.,anti-connexin agents and compositions) in a variety of manners,including for example: (a) by directly affixing to the implant or devicean anti-connexin agent or composition (e.g., by either spraying theimplant or device with a polymer/drug film, or by dipping the implant ordevice into a polymer/drug solution, or by other covalent or noncovalentmeans); (b) by coating the implant or device with a substance such as ahydrogel which will in turn absorb the anti-connexin composition (oranti-connexin factor above); (c) by interweaving anti-connexincomposition coated thread (or the polymer itself formed into a thread)into the implant or device; (d) by inserting the implant or device intoa sleeve or mesh which is comprised of or coated with an anti-connexincomposition; (e) constructing the implant or device itself with ananti-connexin agent or composition; or (f) by otherwise adapting theimplant or device to release the anti-connexin agent, for example byproviding multiple layers or other delivery vehicles, layers or otherdelivery vehicles with differing release characteristics, or bypreparing a device with different parts or regions that include (bylayers or otherwise) different compounds (with or without differentrelease characteristics). Within preferred embodiments of the invention,the composition should firmly adhere to the implant or device duringstorage and at the time of insertion. The anti-connexin agent orcomposition should also preferably not degrade during storage, prior toinsertion, or when warmed to body temperature after insertion inside thebody (if this is required). In addition, it should preferably coat theimplant or device smoothly and evenly, with a uniform distribution ofanti-connexin agent, while not changing the stent contour. Withinpreferred embodiments of the invention, the anti-connexin agent orcomposition should provide a uniform, predictable, prolonged release ofthe anti-connexin factor into the tissue surrounding the implant ordevice once it has been deployed. For vascular stents, in addition tothe above properties, the composition should not render the stentthrombogenic (causing blood clots to form), or cause significantturbulence in blood flow (more than the stent itself would be expectedto cause if it was uncoated).

Delivery of anti-connexin agents utilizing a stent can be carried out ina number of ways, including, for example, from the struts of a stent, astent graft, the catheter used to deliver the stent, the stent cover orsheath. Various methods of applying a therapeutic agent to a stent andadministering the therapeutic agent via the stent are disclosed in U.S.Pat. Nos. 6,702,850; 6,585,764; 6,358,556; 6,344,028; 6,251,136;5,697,967; 5,599,352; 5,591,227; 5,464,650; 5,304,121; 5,163,952;5,092,877; 4,994,071; and 4,916,193, the disclosures of which areincorporated in their entirety herein by reference. Other devices may becoated or impregnated using these or other techniques in the art,including those now known or later developed.

Any one or more of the foregoing medical devices can include anoverlayer of any type, including, for example, a fabric such as asheath, an encasement, a layer, or a coating, such that the fabricoverlayer is in contact with body tissue or fluids such as blood.Alternatively, instead of a fabric overlayer, the medical device mayinclude any other type of layer such as, for example, a mesh, coil,wire, inflatable balloon, bead, sheet, or any other structure which iscapable of being used or implanted at a target location, including, forexample, intravascular target locations, intra-joint locations,intraluminal target locations, intra-orbital and intra-ocular targetlocations, target locations within solid tissue, joints, heart,intestine, eyes, etc.

Impregnated Dressings and Matrices

In one aspect, medical devices are provided in the form of a dressing ormatrix comprising one or more anti-connexin agents, one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents. In certain embodiments, the one or more agents of theinvention are provided in the form of a liquid, semi solid or solidcomposition for application directly, or the composition is applied tothe surface of, or incorporated into, a solid contacting layer such as adressing gauze or matrix. The dressing composition may be provided forexample, in the form of a fluid or a gel. The one or more anti-connexinagents and one or more therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents may be provided incombination with conventional pharmaceutical excipients for topicalapplication. Suitable carriers include: Pluronic gels, Polaxamer gels,Hydrogels containing cellulose derivatives, including hydroxyethylcellulose, hydroxymethyl cellulose, carboxymethyl cellulose,hydroxypropylmethyl cellulose and mixtures thereof; and hydrogelscontaining polyacrylic acid (Carbopols). Suitable carriers also includecreams/ointments used for topical pharmaceutical preparations, e.g.,creams based on cetomacrogol emulsifying ointment. The above carriersmay include alginate (as a thickener or stimulant), preservatives suchas benzyl alcohol, buffers to control pH such as disodium hydrogenphosphate/sodium dihydrogen phosphate, agents to adjust osmolarity suchas sodium chloride, and stabilizers such as EDTA.

Suitable dressings or matrices may include, for example, the followingwith one or more anti-connexin agents with one or more therapeuticagents, agents useful for wound healing and/or gap junction modifyingagents:

1) Absorptives: suitable absorptives may include, for example,absorptive dressings, which can provide, for example, a semi-adherentquality or a non-adherent layer, combined with highly absorptive layersof fibers, such as for example, cellulose, cotton or rayon.Alternatively, absorptives may be used as a primary or secondarydressing.

2) Alginates: suitable alginates include, for example, dressings thatare non-woven, non-adhesive pads and ribbons composed of naturalpolysaccharide fibers or xerogel derived from seaweed. Suitablealginates dressings may, for example, form a moist gel through a processof ion exchange upon contact with exudate. In certain embodiments,alginate dressings are designed to be soft and conformable, easy topack, tuck or apply over irregular-shaped areas. In certain embodiments,alginate dressings may be used with a second dressing.

3) Antimicrobial Dressings: suitable antimicrobial dressings mayinclude, for example, dressings that can facilitate delivery ofbioactive agents, such as, for example, silver and polyhexamethylenebiguanide (PHMB), to maintain efficacy against infection, where this isneeded or desirable. In certain embodiments, suitable antimicrobialdressings may be available as for example, as sponges, impregnated wovengauzes, film dressings, absorptive products, island dressings, nylonfabric, non-adherent barriers, or a combination of materials.

4) Biological & Biosynthetics: suitable biological dressings orbiosynthetic dressings may include, for example, gels, solutions orsemi-permeable sheets derived from a natural source. In certainembodiments, a gel or solution is applied to the treatment site andcovered with a dressing for barrier protection. In another embodiment, asheet is placed in situ which may act as membrane, remaining in placeafter a single application.

5) Collagens: suitable collagen dressings may include, for example,gels, pads, particles, pastes, powders, sheets or solutions derived fromfor example, bovine, porcine or avian sources or other natural sourcesor donors. In certain embodiments, the collagen dressing may interactwith treatment site exudate to form a gel. In certain embodiments,collagen dressing may be used in combination with a secondary dressing.

6) Composites: suitable composite dressings may include, for example,dressings that combine physically distinct components into a singleproduct to provide multiple functions, such as, for example, a bacterialbarrier, absorption and adhesion. In certain embodiment, the compositedressings are comprised of, for example, multiple layers and incorporatea semi- or non-adherent pad. In certain embodiment, the composite mayalso include for example, an adhesive border of non-woven fabric tape ortransparent film. In certain other embodiment, the composite dressingmay function as for example, either a primary or a secondary dressingand in yet another embodiment, the dressing may be used in combinationwith topical pharmaceutical composition.

7) Contact Layers: suitable contact layer dressings may include, forexample, thin, non-adherent sheets placed on an area to protect tissuefrom for example, direct contact with other agents or dressings appliedto the treatment site. In certain embodiments, contact layers may bedeployed to conform to the shape of the area of the treatment site andare porous to allow exudate to pass through for absorption by anoverlying, secondary dressing. In yet another embodiment, the contactlayer dressing may be used in combination with topical pharmaceuticalcomposition. In another embodiment, internal dressing, contact sheets orfilms may be used to reduce or prevent abnormal wound healing in themuscle, connective, epithelial and nerve tissue.

8) Elastic Bandages: suitable elastic bandages may include, for example,dressings that stretch and conform to the body contours. In certainembodiment, the fabric composition may include for example, cotton,polyester, rayon or nylon. In certain other embodiments, the elasticbandage may for example, provide absorption as a second layer ordressing, to hold a cover in place, to apply pressure or to cushion atreatment site.

9) Foams: suitable foam dressings may include, for example, sheets andother shapes of foamed polymer solutions (including polyurethane) withsmall, open cells capable of holding fluids. Exemplary foams may be forexample, impregnated or layered in combination with other materials. Incertain embodiment, the absorption capability may be adjusted based onthe thickness and composition of the foam. In certain other embodiments,the area in contact with the treatment site may be non-adhesive for easyremoval. In yet another embodiment, the foam may be used in combinationwith an adhesive border and/or a transparent film coating that can serveas an anti-infective barrier.

10) Gauzes & Non-Woven dressings: suitable gauze dressings and wovendressings may include, for example, dry woven or non-woven sponges andwraps with varying degrees of absorbency. Exemplary fabric compositionmay include, for example, cotton, polyester or rayon. In certainembodiment, gauzes and non-woven dressing may be available sterile ornon-sterile in bulk and with or without an adhesive border. Exemplarygauze dressings and woven dressings may be used for cleansing, packingand covering a variety of treatment sites.

11) Hydrocolloids: suitable hydrocolloid dressings may include, forexample, wafers, powders or pastes composed of gelatin, pectin orcarboxymethylcellulose. In certain embodiment, wafers are self-adheringand available with or without an adhesive border and in a wide varietyof shapes and sizes. Exemplary hydrocolloids are useful on areas thatrequire contouring. In certain embodiments, powders and pasteshydrocolloids may use used in combination with a secondary dressing.

12) Hydrogels (Amorphous): suitable amorphous hydrogel dressings mayinclude, for example, formulations of water, polymers and otheringredients with no shape, designed to donate moisture and to maintain amoist healing environments and or to rehydrate the treatment site. Incertain embodiment, hydrogels may be used in combination with asecondary dressing cover.

13) Hydrogels: Impregnated Dressings: suitable impregnated hydrogeldressings may include, for example, gauzes and non-woven sponges, ropesand strips saturated with an amorphous hydrogel. Amorphous hydrogels mayinclude for example, formulations of water, polymers and otheringredients with no shape, designed to donate moisture to a drytreatment site and to maintain a moist healing environment.

14) Hydrogel Sheets: suitable hydrogel sheets may include for example,three-dimensional networks of cross-linked hydrophilic polymers that areinsoluble in water and interact with aqueous solutions by swelling.Exemplary hydrogels are highly conformable and permeable and can absorbvarying amounts of drainage, depending on their composition. In certainembodiment, the hydrogel is non-adhesive against the treatment site ortreated for easy removal.

15) Impregnated Dressings: suitable impregnated dressings may include,for example, gauzes and non-woven sponges, ropes and strips saturatedwith a solution, an emulsion, oil, gel or some other pharmaceuticallyactive compound or carrier agent, including for example, saline, oil,zinc salts, petrolatum, xeroform and scarlet red as well as thecompounds described herein.

16) Silicone Gel Sheets: suitable silicone gel sheet dressings mayinclude, for example, soft covers composed of cross-linked polymersreinforced with or bonded to mesh or fabric.

17) Solutions: suitable liquid dressings may include, for example,mixtures of multiprotein material and other elements found in theextracellular matrix. In certain embodiment, exemplary solutions may beapplied to the treatment site after debridement and cleansing and thencovered with an absorbent dressing or a nonadherent pad.

18) Transparent Films: suitable transparent film dressings may includepolymer membranes of varying thickness coated on one side with anadhesive. In certain embodiments, transparent films are impermeable toliquid, water and bacteria but permeable to moisture vapor andatmospheric gases. In certain embodiments, the transparency allowsvisualization of the treatment site.

19) Fillers: suitable filler dressings may include, for example, beads,creams, foams, gels, ointments, pads, pastes, pillows, powders, strandsor other formulations. In certain embodiment, fillers are non-adherentand may include a time-released antimicrobial. Exemplary fillers may beuseful to maintain a moist environment, manage exudate, and fortreatment of for example, partial- and full-thickness wounds, infectedwounds, draining wounds and deep wounds that require packing.

Tissue can be damaged, and inflammation can occur, during the use ofmedical devices during medical procedures, and from the implantation ofmedical devices.

Mammals that may be treated using the described and claimed devices andmethods include, for example, a human being having, or at risk fordeveloping, for example, tissue damage and/or organ dysfunction, forexample cardiovascular, ocular, gastrointestinal, CNS or internal organtissue damage and/or dysfunction, and/or inflammation.

In one aspect of the invention, the medical device comprising ananti-connexin agent, e.g., an anti-connexin 43 agent, is a ventricularassist device. In another aspect of the invention, the medical devicecomprising an anti-connexin agent, e.g., an anti-connexin 43 agent, is aleft ventricular assist device. In yet another aspect of the invention,the medical device comprising an anti-connexin agent, e.g., ananti-connexin 43 agent, is a right ventricular assist device.

In one aspect of the invention, the medical device comprising ananti-connexin agent, e.g., an anti-connexin 43 agent, comprises a stent.In another aspect, the stent comprising an anti-connexin agent, e.g., ananti-connexin 43 agent, is a drug-eluting stent.

In one aspect of the invention, the medical device comprising ananti-connexin agent, e.g., an anti-connexin 43 agent, comprises asuture. In another aspect, the suture comprising an anti-connexin agent,e.g., an anti-connexin 43 agent, is a coated or impregnated drug-elutingsuture.

In another aspect of the invention, a medical device comprises aninstrument, such as a catheter, for example, or an implant suitable forintroduction into a subject of which at least a portion comprises ananti-connexin agent, e.g., an anti-connexin 43 agent, that is availableto ameliorate gap junction formation and/or hemichannel opening in thesubject when the device is used.

In one embodiment, the release rate of the anti-connexin agent iscontrolled. In one embodiment of the invention, the anti-connexin agentmay be released slowly or over a sustained period. In one embodiment theanti-connexin agent is released, for example, over the period of theresorption or degradation of the body of the medical device, or aportion thereof.

In one embodiment of the invention, the surface of the medical devicecomprises an anti-connexin agent, e.g., an anti-connexin 43 agent. Inone aspect of the invention an anti-connexin agent is bound, eitherdirectly or indirectly, to a medical device. In another aspect of theinvention, an anti-connexin agent is bound, directly or indirectly, to asurface of a medical device. In another aspect of the invention, thissurface contacts a tissue within a subject. In one embodiment, thetarget tissue is heart tissue, vascular tissue, muscle tissue, orconnective tissue. In yet another embodiment, this surface contacts asite of injury or potential injury. In another embodiment, the medicaldevice provides for surface contact release of the anti-connexin agent,e.g., an anti-connexin 43 agent.

In one embodiment, an anti-connexin agent, e.g., an anti-connexin 43agent, is present in a coating on a surface of the medical device. Suchcoatings include, for example, synthetic or natural matrices, forexample, fibrin or acetate-based polymers, mixtures of polymers orcopolymers, which can also be bioresorbable or biodegradable matrices,and which matrices have or include or incorporate an anti-connexinagent. Such matrices can, for example, provide for surface contact ormetered or sustained release of one or more anti-connexin agent.

In another embodiment, the device comprising an anti-connexin agent,e.g., an anti-connexin 43 agent, may be formed, at least in part, forexample, from a biodegradable or bioresorbable polymer material. Polymermaterials can include, for example, but are not limited to, nylon,polyethylene perthalate, polytetrafluoroethylene, etc. Other polymersmay also include, for example, hexafluoropropylene (HFP),tetrafluoroethylene (TFE), vinylidenefluoride,1-hydropentafluoropropylene, perfluoro(methyl vinyl ether),chlorotrifluoroethylene (CTFE), pentafluoropropene, trifluoroethylene,hexafluoroacetone and hexafluoroisobutylene.

In another aspect, the surface of a medical device comprising ananti-connexin agent, e.g., an anti-connexin 43 agent, may be composed oforganic materials or a composite of organic and inorganic materials.Examples of such materials include, but are not limited to, for example,synthetic polymers or copolymers containing one or more anti-connexinagents, surfaces upon which a functionalized monolayer containing theanti-connexin agent is adsorbed or otherwise attached, or syntheticpolymeric materials or proteins blended with one or more anti-connexinagents.

In another aspect, all or a portion of the medical device is coated withan anti-connexin agent, e.g., an anti-connexin 43 agent, either as thecoating per se or in a coating matrix, for example; or all or a portionof the medical device may be produced from a material which includes ananti-connexin agent, for example, a polymer which has admixed therewithan anti-connexin agent or which includes a functionalized anti-connexinagent; or all or a portion of the tissue-contacting surfaces of themedical device may be derivatized with an anti-connexin agent.

The medical device can be coated using any one or more methods known inthe art, for example, dip coating, spray coating, sponging or brushing.The coating may contain the anti-connexin agent, e.g., an anti-connexin43 agent, in a weight percentage range of from about 0.0001% to about30%, for example, although other amounts are contemplated and may beused. Thus, according to one option, the coating may containanti-connexin agent in a weight percentage range of about 0.001% toabout 25%, alternatively in a range of about 0.01% to about 20%, about0.1% to about 15%, about 0.5% to about 12%, about 1% to about 10%, about2% to about 10%, about 5% to about 10%, about 0.01% to about 5%, about0.1% to about 5% or about 0.5% to about 5%. The weight percentage forthe anti-connexin agent will be adjusted as appropriate, in view ofconsiderations which include, but are not limited to, the following: thedose of anti-connexin agent to be delivered locally, the rate of releaseof anti-connexin agent from the coating and the time period for deliveryof anti-connexin agent. Included within the coating may be furthersuitable excipients, for example, a polymer and/or those excipients thataid in the binding of the coating to the anti-connexin agent (or visaversa) and/or that aid in the release of the anti-connexin agent.Alternatively, excipients can be bound to the anti-connexin agent, forexample, a polymer or other substance that entraps the anti-connexinagent on or within the surface of the medical device.

The medical device can also be coated using any one or more methodsknown in the art, to deliver amounts of an anti-connexin agent rangingfrom about 0.1 micrograms to about 1 milligram or more, in amountsranging from about 1 micrograms to about 750 micrograms, in amountsranging from about 5 micrograms to about 600 micrograms, in amountsranging from about 10 micrograms to about 500 micrograms, in amountsranging from about 20 micrograms to about 400 micrograms, in amountsranging from about 30 micrograms to about 300 micrograms, in amountsranging from about 50 micrograms to about 200 micrograms, and in amountsranging from about 50 micrograms to about 100 micrograms, for example,although other amounts are contemplated and may be used, includingranges that include only the lower amounts (or the upper amounts) fromthe above ranges, for example, amounts ranging from about 1-10micrograms, about 1-5 micrograms, about 10-50 micrograms, and so on (andamounts ranging from about 100-200 micrograms, about 500-600 micrograms,about 300-400 micrograms, and so on). The amounts of the anti-connexinagent will be adjusted as appropriate, in view of considerations thatinclude, but are not limited to, the following: the dose ofanti-connexin agent to be delivered locally, the rate of release ofanti-connexin agent from the coating and the time period for delivery ofanti-connexin agent, the size and nature of the injury, the extent ofanticipated tissue damage, inflammation, and so on.

In certain embodiments, methods or coating and dosing/dosage informationfor the exemplary compounds useful in accordance with the presentdisclosure may be found in U.S. Pat. No. 7,153,822 to Jensen et al.,U.S. Pat. No. 7,250,397, Green et al., WO2006134494; Gourdie et al., seeWO2006069181, and Tudor et al., see WO2003032964.

In another aspect, all or a portion or portions of the medical devicemay be coated with the anti-connexin agent per se or with apharmaceutically acceptable carrier or excipient comprising theanti-connexin agent which serves as a coat or coating matrix. This maybe a solid, liquid, gel or semisolid consistency, for example.

The carrier or matrix can be made of or include agents which provide,for example, for metered or slow or sustained release of theanti-connexin agent and/or other therapeutic agent(s). The coating, forexample, can include albumin that can be either, for example, human orbovine, including humanized bovine serum albumin.

A coating may be applied as a single coating or in multiple coatings orlayers. The multiple coatings or layers can include varying ratios ofanti-connexin agent-to-carrier to vary the release rate of the drug overtime. The multiple coatings or layers can also include differentmedicaments in accordance with a desired treatment plan. In oneembodiment, the coating comprises a plurality of coatings or layers of apolymer/anti-connexin agent mixture, e.g., an anti-connexin 43 agentmixture, applied to the medical device. In another embodiment, theanti-connexin agent-to-polymer ratio in the plurality of coatings orlayers varies.

In another aspect, anti-connexin agents, e.g., an anti-connexin 43agents, may be impregnated or otherwise incorporated into the body ofall or a portion of the medical device.

Additionally, the anti-connexin agent, e.g., an anti-connexin 43 agent,may be provided in one or more reservoirs or channels formed in themedical device, optionally with a coating or membrane of biocompatiblematerial applied over the medical device to control diffusion of thedrug from the reservoirs/channels to the tissue. In one embodiment, themedical device comprises at least one channel formed in an outer surfacethereof, and wherein the anti-connexin agent is included on and/orwithin at least one channel.

In another aspect, if a “burst effect” is desired, the anti-connexinagent, e.g., an anti-connexin 43 agent, may be applied at the outerlayer of the medical device (and/or within the device, as well) so thatan initial amount of the anti-connexin agent is promptly released whenit comes into contact with tissue. Remaining amounts, if any, ofanti-connexin agent included in the inner layers will be released overtime as the anti-connexin agent diffuses through the material. Theanti-connexin agent may be provided either in liquid or solid form.

In another aspect, the coating applied to the medical device can be“recharged”, for example, by way of a catheter or other tubing capableof infusing an anti-connexin agent donor to a previously coated surfaceor impregnated or other device comprising an anti-connexin agent. Forexample, in one embodiment, the anti-connexin agent may befunctionalized to form an anti-connexin agent-protein that will losepotency in vivo as the functionalized-anti-connexin agent-protein ismetabolized, leaving un-derivatized protein. The surface coating can be“recharged” by infusing an anti-connexin agent or anti-connexin agentdonor capable of binding the un-derivatized protein.

In another aspect of the invention, the derivatization of an artificialsurface with an anti-connexin agent provides for the amelioration oftissue damage, and/or enhancement of tissue repair, as well as theprevention and/or amelioration of imflammation. The artificial surfacesmay be composed of organic materials or a composite of organic andinorganic materials. Examples of such materials include syntheticpolymers or copolymers containing one or more anti-connexin agents,surfaces upon which a functionalized monolayer containing theanti-connexin agent is absorbed, or synthetic polymeric materials orproteins which are blended with the anti-connexin agent.

In one embodiment of the invention, the localized and/or time-relatedpresence of an anti-connexin agent, e.g., an anti-connexin 43 agent,administered in a physiologically effective form is efficacious indiminishing, deterring or preventing tissue damage, includinginflammation, after or as a result of instrumental intervention, such asangioplasty, catheterization, or the introduction of a stent (e.g., aPalmaz-Schatz stent), shunt, mesh, joint replacement, or other surgicalor indwelling medical device. Local administration of a stableanti-connexin agent inhibits neutrophil and/or macrophage migrationfollowing vascular arterial balloon injury, for example, as well asinflammation, swelling and hypeplasia. This strategy for the localdelivery of an anti-connexin agent is useful, among other things, forthe treatment of vascular injury following angioplasty, and othertreatments that can result in vessel or tissue disturbance.

In a further embodiment, the invention provides for the localized use ofa functionalized anti-connexin agent-polypeptide or polynucleotide,particularly those which do not elicit any significant or otherwiseundesired immune response. Such functionalized anti-connexinagent-polypeptides or polynucleotides, such as, for example,functionalized anti-connexin agent-albumins, can be present as polymericchains or three dimensional aggregates where the functionalizedanti-connexin agent-polypeptide or polynucleotide is the monomeric unit.The polypeptide or polynucleotide of the monomeric unit can also be usedto aid, for example, in localization. The aggregates may be multipleinter-adherent monomeric units which can optionally be linked bydisulfide bridges or —O— linkages, for example. Additionally, deviceswhich have been substituted or coated with one or more functionalizedanti-connexin agent polypeptides or polynucleotides may be dried andstored.

In another aspect, the invention relates to a method of preventingand/or treating damage, including inflammation, associated with the useor implantation of a medical device in a subject comprising introducinginto said subject a medical device of which at least a portion comprisesan anti-connexin agent, wherein said damage and/or inflammation isprevented, reduce, ameliorated and/or delayed.

In another aspect, the invention related to a method of preventingand/or treating damage and/or inflammation associated with the use orimplantation of a medical device in a subject comprising use orimplantation of a medical device which comprises an anti-connexin agentthat is releasable at its point of contact, wherein the damage and/orinflammation is prevented, amerliorated and/or delayed.

In yet another aspect, the invention relates to a method of preventingand/or treating adverse effects associated with the use or insertion ofa medical device in a subject, wherein an anti-connexin agent is locallyadministered at the site of contact of said medical device, before,during and/or after said use or insertion, wherein said adverse effectsare prevented, ameliorated, reduced or delayed. Such adverse effectsinclude inflammation, swelling, hyperplasia, neutrophil migration, andmacrophage migration, as well as blood vessel disturbance and/orleakage.

In another aspect, the invention relates to a method of treating adamaged vessel in a subject in need thereof which comprises introducinginto said vessel at the site of damage a catheter, a balloon, a graft, amesh, a stent or a shunt comprising an anti-connexin agent, e.g., ananti-connexin 43 agent.

In another aspect, the invention comprises a method of amelioratingtissue damage and/or enhancing tissue repair by locally administeringone or more anti-connexin agents, e.g., an anti-connexin 43 agent, tothe site of tissue in need thereof. Such tissue may be damaged, or havebeen damaged, for example, as a result of the use of a medical device inan invasive procedure. Thus, for example, in treating blockedvasculature, by, for example, angioplasty, damage to the blood vesselcan result. Additionally, tissue damage can result when medical devicesare left within a subject for an extended period of time. Such damagemay be treated by use of an anti-connexin agent. In addition toameliorating tissue damage and/or enhancing repair of the damagedtissue, such treatment can also be used to prevent and/or alleviateand/or delay occlusions, including for example, acute occlusion andreocclusions. Treatment may also be used to prevent and/or ameliorateand/or delay thrombosis and restenosis. Treatment may also be used toprevent and/or ameliorate and/or delay inflammation. Treatment may alsobe used to prevent and/or ameliorate and/or delay hyperplasia. Treatmentmay also be used to prevent and/or ameliorate and/or delay oxidativedamage, including, for example oxidative damage by free radicals,including superoxide damage. In other aspect, the invention, treatmentmay also be used to treat and/or prevent and/or alleviate and/or delayatherosclerotic lesions, and undesired cell proliferation and migration.

In one embodiment the invention comprises a method of amelioratingtissue damage and/or enhancing tissue repair wherein the subject is amammal. In another embodiment the invention comprises a method ofameliorating tissue damage and/or enhancing tissue repair wherein thesubject is human. In another embodiment the invention comprises a methodof ameliorating tissue damage and/or enhancing tissue repair wherein thesubject is selected from the group consisting of domestic and petanimals (for example, horses, dogs and cats), sports animals (forexample, horses and dogs), farm animals, and zoo animals, as well asbirds.

In one embodiment the medical device comprising an anti-connexin agentcan include one or more other therapeutic agents and/or wound healingagents. In one embodiment, the therapeutic agents may be applied orincluded directly with the anti-connexin agent, e.g., an anti-connexin43 agent. In another embodiment, the therapeutic agent may be applied orincluded in the same coating or coating layer with the anti-connexinagent. In yet another embodiment, the therapeutic agent may be appliedor included in separate coating or coating layer or a separate portionof the device.

In one embodiment, the anti-connexin agent is an anti-connexin 43compound. In another embodiment, the anti-connexin agent downregulatesor inhibits connexin 43 mRNA. In another embodiment, the anti-connexinagent suppresses the formation of connexin 43 gap junctions. In anotherembodiment, the anti-connexin agent suppresses the joining of connexin43 hemichannels to form gap junctions. In another embodiment, theanti-connexin agent suppresses connexin 43 hemichannel opening.

In another embodiment, the anti-connexin agent targets connexin 31,connexin 31.1, or connexin 43. Preferably, the anti-connexin agenttargets connexin 43.

There are various techniques known in the art for the localized deliveryof therapeutic agents, for example, but not limited to, by means of asmall catheter which extends from the exterior of the subject to theinternal tissue site, with a mechanical delivery system being providedto administer a therapeutic agent in a continuous or periodic controlleddosage. This method can be utilized for subjects in need of therapeutictreatment by repeat application. Anti-connexin agents, e.g., ananti-connexin 43 agents, can also be delivered arthoscopically usingdevices known in the art, which may themselves be coated or impregnated,for example, with an anti-connexin agent, e.g., an anti-connexin 43agent.

Any catheter, however, is within the scope of the invention, includingthose for the delivery of therapeutic agents or otherwise. Theseinclude, for example, urological catheters (including intermittentcatheters, external catheters, and Foley catheters), pancreaticcatheters, hepatic catheters, infusion catheters, cardiovascularcatheters, renal catheters, hemodynamic monitoring catheters,neurological catheters, and so on. Cannulae, as noted above, are alsoincluded.

The method of applying one or more anti-connexin agents, e.g., ananti-connexin 43 agent, to an internal tissue site of a subjectcomprises, for example, advancing an elongate member, such as a catheter(which may itself be coated or impregnated with one or moreanti-connexin agents), internally into the subject to cause a portion ofthe elongated member to occupy the internal tissue site. A portion ofthe elongated member comprises a lateral wall section which carries theanti-connexin agent in a manner permitting release thereof from thelateral wall section at the internal tissue site once the site has beenreached. This can be determined by the usual methods, such as forexample, fiber optic television, x-ray, etc.

The release of the anti-connexin agent at the internal tissue site canoccur for example by the use of a catheter balloon, which when inflatedcauses the anti-connexin agent to be pressed into and/or onto the tissueat the internal tissue site. This allows at least some of theanti-connexin agent to be retained at the tissue site once the catheterballoon is deflated.

The anti-connexin agent can also be mixed with a controlled releasecarrier and administered in the manner discussed above, for example.Such controlled release carriers can be biodegradable over a period oftime, for example, over a period of hours or days, so that as thecontrolled release carriers are brought into contact with the tissuesand/or fluids the controlled release carriers degrade over time to allowfor a relatively slow, controlled diffusion of the anti-connexin agentto the tissue and/or fluids. The carrier over time will be removed bynatural bodily processes.

The catheter or balloon catheter or other device used to apply a stentto the coronary artery or elsewhere may also be provided with a coatingof heparin or other anti-thrombogenic agent, for example, in conjunctionwith one or more anti-connexin agents, e.g., an anti-connexin 43 agent,that may be combined with a carrier or a controlled release carrier forthe anti-connexin agent. Thus, simultaneously with the application ofthe anti-connexin agent, the heparin or other anti-thrombogenic agent,for example, is applied to the internal tissue site for the long termsuppression of thrombogenic activity in the vicinity of the stent inaddition to therapeutic effects from the anti-connexin agent.

In addition, while the catheter or other elongate member, for example,is being used to position the stent and is being advanced to theinternal tissue site, the portion of the catheter carrying theanti-connexin agent may be enclosed in a protective sheath. The sheathis used to prevent removal of substantial amounts of the anti-connexinagent from the catheter before reaching the desired internal site. Whenthe site is reached, the protective sheath may be withdrawn to exposethe catheter portion carrying the anti-connexin agent. The anti-connexinagent, e.g., an anti-connexin 43 agent, can then be applied to theinternal site, for example, by expansion of a catheter balloon uponwhich the anti-connexin agent resides, or by other processes such asspontaneous dispersion off the catheter into the tissues. If desired,the protective sheath may be a conventional introducer catheter, or itmay be a split introducer sheath to facilitate removal of the sheathfrom the catheter after its withdrawal, for example.

One example of a method of coating a medical device having more than onesurface or requiring only a portion of a surface of the medical deviceto be treated is to treat the device with gas plasma that may, forexample, be composed of a molecular species containing the anti-connexinagent. In the case of stents, it is particularly desirable to treat theentire surface. In the case of balloons mounted on catheters, it isdesirable to coat at least the outer cylindrical surface of the balloonthat will be in contact with a blood vessel or other tissue when theballoon is inflated.

Alternatively, the anti-connexin agent can be mixed with polymers (bothdegradable and non degradable), for example, to hold a anti-connexinagent(s) to a stent or graft or other device, or the anti-connexin agentcan be entrapped into the material of, for example, a stent or graftbody, or other device.

Alternatively the anti-connexin agent can be covalently bound to, forexample, a stent or other device via solution chemistry techniques ordry chemistry techniques (for example, vapour deposition methods such asrf-plasma polymerization) and combinations thereof.

Another method of coating a surface of a medical device with one or moreanti-connexin agents, e.g., an anti-connexin 43 agent, comprisescontacting the surface with an anti-connexin agent(s) so as to cause thesurface to be coated with the particular anti-connexin agent. Coating ofthe artificial surface may be accomplished using the methods describedin the Examples, or other methods known in the art.

For example, coating a surface with an anti-connexin agent, e.g., ananti-connexin 43 agent, can be achieved by bathing the artificialsurface, either by itself or within a device, in a solution containingthe anti-connexin agent. In addition, synthetic anti-connexin agents maybe coated onto an artificial surface by a variety of chemical techniqueswhich are well known in the art. Such techniques include attaching theanti-connexin agent, for example, a functionalized compound, by means ofa linking group, or to a nucleophilic center, epoxide, lactone, analpha- or beta-saturated carbon chain, alkyl halide, carbonyl group, orSchiff base, by way of a reactive group, for example, a free thiol.

A medical device may be coated using a variety of different techniques.The coating may be applied as a mixture, solution or suspension ofpolymeric material, for example, and one or more finely dividedanti-connexin agents dispersed in an organic vehicle or a solution orpartial solution of such anti-connexin agent(s) or binding compound(s)in a solvent or vehicle for the polymer and/or anti-connexin agent(s) orbinding compound(s). For the purposes of this patent, the term “finelydivided” means any type or size of included material from dissolvedmolecules through suspensions, colloids and particulate mixtures, orthat otherwise serve the intended purpose. One or more anti-connexinagents can be disbursed in the carrier material, which may be thepolymer, a solvent, or both, for example. The coating may be applied asa single layer or as a plurality of layers, typically relatively thinlayers, sequentially applied, for example, in relatively rapid sequence.In some applications, the coating may further be characterized as acomposite initial tie coat, or undercoat, and a composite topcoat. Thecoating thickness ratio of the topcoat to the undercoat may vary withthe desired effect and/or the elution system. Typically, the topcoat andundercoat are of different formulations, but need not be. Providing ananti-connexin agent(s) and/or binding compound(s) as a plurality oflayers on the medical device enables both an initial burst effect ofdrug elution and the drug release kinetic profile associated with longterm therapeutic effect to be controlled.

Various combinations of coating materials, such as polymer coatingmaterials, for example, can be coordinated with biologically orchemically active species of interest to produce desired effects whencoated on stents or other medical devices to be implanted, or inserted,in accordance with the invention. Loadings of therapeutic materials mayvary, as well as the types of therapeutic material. The mechanism ofincorporation of the biologically or chemically active species into thesurface coating, as well as the egress mechanism, depends both on thenature of the surface coating polymer and the therapeutic material to beincorporated. The mechanism of release also depends on the mode ofincorporation. The therapeutic material may elute via interparticlepaths or be administered via transport or diffusion through theencapsulating material itself.

Suitable polymers for use in the coating include, for example, a polymerthat is biocompatible and minimizes irritation to the vessel wall when amedical device is implanted. It is advantagous that such polymer exhibithigh elasticity/ductility, resistance to erosion, elasticity, andcontrolled drug release. Such polymer may be either a biostable or abioabsorbable polymer depending on the desired rate of release or thedesired degree of polymer stability. Bioabsorbable polymers that may beused include poly(L-lactic acid), polycaprolactone,poly(lactide-co-glycolide), poly(ethylene-vinyl acetate),poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoester,polyanhydride, poly(glycolic acid), poly(D,L-lactic acid), poly(glycolicacid-co-trimethylene carbonate), polyphosphoester, polyphosphoesterurethane, poly(amino acids), cyanoacrylates, poly(trimethylenecarbonate), poly(iminocarbonate), copoly(ether-esters) (e.g. PEO/PLA),polyalkylene oxalates, polyphosphazenes and biomolecules such as fibrin,fibrinogen, cellulose, starch, collagen and hyaluronic acid.

Also, biostable polymers with a relatively low chronic tissue responsesuch as polyurethanes, silicones, and polyesters could be used and otherpolymers may also be used if they can be dissolved and cured orpolymerized on the medical device such as polyolefins, polyisobutyleneand ethylene-alphaolefin copolymers; acrylic polymers and copolymers,ethylene-co-vinylacetate, polybutylmethacrylate, vinyl halide polymersand copolymers, such as polyvinyl chloride; polyvinyl ethers, such aspolyvinyl methyl ether; polyvinylidene halides, such as polyvinylidenefluoride and polyvinylidene chloride; polyacrylonitrile, polyvinylketones; polyvinyl aromatics, such as polystyrene, polyvinyl esters,such as polyvinyl acetate; copolymers of vinyl monomers with each otherand olefins, such as ethylene-methyl methacrylate copolymers,acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetatecopolymers; polyamides, such as Nylon 66 and polycaprolactam; alkydresins; polycarbonates; polyoxymethylenes; polyimides; polyethers; epoxyresins, polyurethanes; rayon; rayon-triacetate; cellulose, celluloseacetate, cellulose butyrate; cellulose acetate butyrate; cellophane;cellulose nitrate; cellulose propionate; cellulose ethers; andcarboxymethyl cellulose.

Accordingly, the coating comprising an anti-connexin agent, e.g., ananti-connexin 43 agent, may be formed, at least in part, for example,from a biodegradable or bioresorbable polymer material. Polymermaterials can include, for example, but are not limited to, nylon,polyethylene perthalate, polytetrafluoroethylene, etc. Other polymersmay also include, for example, hexafluoropropylene (HFP),tetrafluoroethylene (TFE), vinylidenefluoride,1-hydropentafluoropropylene, perfluoro(methyl vinyl ether),chlorotrifluoroethylene (CTFE), pentafluoropropene, trifluoroethylene,hexafluoroacetone and hexafluoroisobutylene.

The desired release rate profile can be tailored by, for example,varying the coating thickness, the radial distribution (layer to layer)of bioactive materials, the number of layers, the mixing method, and theamount of bioactive material(s), the combination of different materials,for example, matrix polymer materials, at different layers, and thecrosslink density of a polymeric material. The crosslink density isrelated to the amount of crosslinking which takes place and also therelative tightness of the matrix created by the particular crosslinkingagent used. Thus, the curing process of such a coating typicallydetermines the amount of crosslinking and also the crosslink density ofthe polymer material. For bioactive materials released from acrosslinked matrix, a crosslink structure of greater density willincrease release time and reduce burst effect. By applying at least onetherapeutically active anti-connexin agent at the outer layer of themedical device, a burst effect may be made to occur where a large amountof the anti-connexin agent is immediately or promptly released when itcomes into contact with the tissue. Subsequently, longer term release ofthe anti-connexin agent will occur as it diffuses through the material,for example, a polymeric material.

The elution kinetics of an anti-connexin agent, e.g., an anti-connexin43 agent, can be modified to meet the needs of the particular medicaldevice application. For example, medical devices can be coated using acombination of an anti-connexin agent with one, two or more othermedicaments, where the release sequence can be rate controlled. Forexample, one or more anti-connexin agents may be combined in theundercoat layer, and anti-thrombotic drugs, for example, heparin, may beprovided in the topcoat layer. In this manner, the anti-thrombotic drugswill elute first, followed by the anti-connexin agent(s). In the casewhere the medical device is an implanted stent, this combination ofdrugs may better enable safe encapsulation of the implanted stent. Anydesired drug may be included in this manner.

Alternately, the drug coating may include a base coat layer applieddirectly to the surfaces of the medical device, a second layer whichincludes a pharmacological agent, for example, an anti-connexin agent,e.g., an anti-connexin 43 agent, and a third layer in the form of acontinuous membrane encapsulating the entire device. The base coatserves as a primer by readily adhering to the surface of the medicaldevice and then readily accepting and retaining the anti-connexinagent(s) applied thereto. The base coat may include materials such asvitronectin, fibronectin, gelatin, collagen, and/or other similarmaterials, for example, which are relatively inexpensive and dry to forma sticky coating. An anti-connexin agent may be supplied in the form ofdry, micronized particles, for example, that readily adhere to thesticky base layer surface. It is preferred that the anti-connexin agentmay have a particle size of about 0.005 to about 3.0 micro metres, orsuch size may be different, if desired. Other particle sizes arecontemplated depending on the particular medical application and deviceto which the anti-connexin agent(s) is/are being applied. The outermembrane or layer may encapsulate the entire medical device to cover allof its surfaces, including any bare device structure, any exposed basecoating or the layer of micronized anti-connexin agent(s) or othermedicament particles.

The material selected to form the membrane is dependent on its membraneforming characteristics and its biocompatibility, as well as itspermeability to an anti-connexin agent, e.g., an anti-connexin 43 agent.The chemical composition of the membrane forming polymer, for example,and that of an anti-connexin agent, in combination with the thickness ofthe applied outer layer, will determine the diffusion rate of theanti-connexin agent.

The overall coating should be thin enough so that it will notsignificantly increase the profile of the medical device when insertedinto a mammal. In the case of implantable stents, the coating is, forexample, from between 0.005 microns to about 400 microns thick. However,other thicknesses may be utilized without departing from the spirit andscope of the present invention. The adhesion of the coating and the rateat which the drug is delivered can be controlled by the selection of anappropriate bioabsorbable or biostable material, such as a polymer, andby the ratio of the drug-to-polymer in the solution. In the case wheremultiple layers are utilized to coat the medical device, the releaserate can be further controlled by varying the ratio of, for example,anti-connexin agent-to-polymer, in the multiple layers. For example, ahigher anti-connexin agent-to-polymer ratio in the outer layers than inthe inner layers would result in a higher early dose which woulddecrease over time.

In an alternate form, the medical device can include reservoirs, orchannels, which may be loaded with one or more anti-connexin agents,e.g., an anti-connexin 43 agent. Such reservoirs can aid in decreasingthe profile of the coated device, since the anti-connexin agent or aportion of the anti-connexin agent would be provided within thereservoir or channel. In such an embodiment, the anti-connexin agent isprovided in the reservoirs, and a coating or membrane of biocompatiblematerial is applied over the reservoir which controls the diffusion ofthe anti-connexin agent from the reservoirs to the tissue. Furtherlayers of anti-connexin agent and/or materials, for example, polymericmaterials, may be applied to the device in accordance with the teachingsherein without departing from the spirit and scope of the presentinvention.

The amount of anti-connexin agent included in the layer(s) will varydepending on the dosage required for effective therapeutic treatment. Atherapeutically effective amount of a anti-connexin agent may bedetermined from the doses of such compounds administered topically thattypically will vary from about 0.1 micrograms to about 1 milligram ormore per square millimeter or per square centimeter of device surface,in amounts ranging from about 1 micrograms to about 750 micrograms, inamounts ranging from about 5 micrograms to about 600 micrograms, inamounts ranging from about 10 micrograms to about 500 micrograms, inamounts ranging from about 20 micrograms to about 400 micrograms, inamounts ranging from about 30 micrograms to about 300 micrograms, inamounts ranging from about 50 micrograms to about 200 micrograms, and inamounts ranging from about 50 micrograms to about 100 micrograms, forexample, although other amounts are contemplated and may be used,including ranges that include only the lower amounts (or the upperamounts) from the above ranges, for example, amounts ranging from about1-10 micrograms, about 1-5 micrograms, about 10-50 micrograms, and so on(and amounts ranging from about 100-200 micrograms, about 500-600micrograms, about 300-400 micrograms, and so on). The amounts of theanti-connexin agent will be adjusted as appropriate, in view ofconsiderations that include, but are not limited to, the following: thedose of anti-connexin agent to be delivered locally, the rate of releaseof anti-connexin agent from the coating and the time period for deliveryof anti-connexin agent, the size and nature of the injury, the extent ofanticipated tissue damage, inflammation, and so on.

The amount of anti-connexin agent in the coating or layer is adjusted sothat the desired dose of anti-connexin agent, e.g., an anti-connexin 43agent, is delivered at the desired delivery rate for the desired time ofdelivery. The time of delivery will depend on factors which include thetime period for which the device is intended to be implanted in thesubject.

In one embodiment the coating may contain anti-connexin agent in aweight percentage of from about 0.0001% to about 30%. As noted above,depending on dose, rate of delivery, period of delivery and otherfactors, other amounts are contemplated and may be used. Thus, accordingto one option, the coating may contain anti-connexin agent in a weightpercentage range of about 0.001% to about 25%, alternatively in a rangeof about 0.01% to about 20%, about 0.1% to about 15%, about 0.5% toabout 12%, about 1% to about 10%, about 2% to about 10%, about 5% toabout 10%, about 0.01% to about 5%, about 0.1% to about 5% or about 0.5%to about 5%. The weight percentage for the anti-connexin agent will beadjusted as appropriate, in view of considerations which include, butare not limited to, the following: the dose of anti-connexin agent to bedelivered locally, the rate of release of anti-connexin agent from thecoating and the time period for delivery of anti-connexin agent.

Other therapeutically effective dosage ranges may be useful. One skilledin the art can customize the desired rate and/or dosage of anti-connexinagent delivery by evaluation and/or selection of an appropriatebioabsorbable or biostable polymer and by the ratio of anti-connexinagent-to-polymer in the coating, for example.

The particular surface or surfaces on which the anti-connexin agent isdeposited determines where the anti-connexin agent will be deliveredupon implantation. For example, in the case of a stent, anti-connexinagent deposited on the outer exterior surfaces of the stent will causethe anti-connexin agent to pass directly into the lumen wall, whiledeposition of the anti-connexin agent on the outer interior surfaces ofthe stent will cause the anti-connexin agent to be released directlyinto the blood stream. Alternately, coating only the upstream edge oronly the downstream edge of the stent may be desirable to achieve adesired effect. By selectively coating the stent, or other medicaldevice, surfaces with the anti-connexin agent or other medicaments, thedistribution of the anti-connexin agent may be precisely controlled.

It is also contemplated that artificial surfaces will vary depending onthe nature of the surface, and such characteristics as contour,crystallinity, hydrophobicity, hydrophilicity, capacity for hydrogenbonding, and flexibility of the molecular backbone and polymers.Therefore, using routine methods, one of ordinary skill will be able tocustomize the coating technique by adjusting such parameters as theamount of anti-connexin agent, length of treatment, temperature,diluents, and storage conditions, in order to provide optimal coating ofeach particular type of surface.

After the device or artificial material has been coated or impregnatedwith one or more anti-connexin agents, it will be suitable for itsintended use, for example, implantation as a heart valve, insertion as acatheter, or insertion as a stent, and so on. The coated device orartificial surface will be suitable for use in conjunction with ananimal, generally mammals, including humans.

Another embodiment of an anti-connexin agent pertains to thederivatization of synthetically derived polymeric materials byattachment of a functionalized anti-connexin agent such as, for example,a functionalized compound as described herein.

Dosage Forms and Formulations

A therapeutically effective amount of each of the combination partners(e.g. an anti-connexin agent and a wound healing agent) may beadministered simultaneously, separately or sequentially and in anyorder. The agents may be administered separately or as a fixedcombination. When not administered as a fixed combination, preferredmethods include the sequential administration of one or moreanti-connexin agents and one or more agents useful for wound healing,either or both of which are provided in amounts or doses that are lessthat those used when the agent or agents are administered alone, i.e.,when they are not administered in combination, either physically or inthe course of treatment of a wound. Such lesser amounts of agentsadministered are typically from about one-twentieth to about one-tenththe amount or amounts of the agent when administered alone, and may beabout one-eighth the amount, about one-sixth the amount, about one-fifththe amount, about one-fourth the amount, about one-third the amount, andabout one-half the amount when administered alone. Preferably, theagents are administered sequentially within at least about one-half hourof each other. The agents may also be administered with about one hourof each other, with about one day to about one week of each other, or asotherwise deemed appropriate. Preferably, the anti-connexin agent isadministered first. Preferably, where one or more anti-connexin agentsare used, an anti-connexin peptide or anti-connexin peptidomimetic,e.g., an anti-connexin agent that can block or reduce hemichannelopening, is administered prior to the administration of an anti-connexinagent that blocks or reduce connexin expression or the formation ofhemichannels or gap junctions, e.g., by downregulation of connexinprotein expression. Preferably, the anti-connexin agent or agents is/areanti-connexin 43 agent(s).

The agents of the invention of the may be administered via a medicaldevice to a subject in need of treatment, such as a subject with any ofthe diseases or conditions mentioned herein. The condition of thesubject can thus be improved. The anti-connexin agent and combinationalpartner may thus be used in the treatment of the subject's body bytherapy using the device. They may be used in the manufacture of amedical device as mentioned herein. Thus, in accordance with theinvention, there are provided formulations by which cell-cellcommunication can be downregulated in a transient and site-specificmanner.

The anti-connexin agent may be present in a substantially isolated form.It will be understood that the product may be mixed with carriers ordiluents which will not interfere with the intended purpose of theproduct and still be regarded as substantially isolated. A product ofthe invention may also be in a substantially purified form, in whichcase it will generally comprise about 90%, e.g. at least about 95%, atleast about 98% or at least about 99% of the polynucleotide (or otheranti-connexin agent) or dry mass of the preparation.

Depending on the intended route of application, the products of theinvention may, for example, take the form of solutions, suspensions,instillations, salves, creams, gels, foams, ointments, emulsions,lotions, paints, sustained release formulations, or powders, andtypically contain about 0.1%-95% of active ingredient(s), preferablyabout 0.2%-70% as applied to medical devices. Other suitableformulations include pluronic gel-based formulations,carboxymethylcellulose(CMC)-based formulations, andhyroxypropylmethylcellulose(HPMC)-based formulations. Other usefulformulations include slow or delayed release preparations.

Gels or jellies may be produced using a suitable gelling agentincluding, but not limited to, gelatin, tragacanth, or a cellulosederivative and may include glycerol as a humectant, emollient, andpreservative. Ointments are semi-solid preparations that consist of theactive ingredient incorporated into a fatty, waxy, or synthetic base.Examples of suitable creams include, but are not limited to,water-in-oil and oil-in-water emulsions. Water-in-oil creams may beformulated by using a suitable emulsifying agent with propertiessimilar, but not limited, to those of the fatty alcohols such as cetylalcohol or cetostearyl alcohol and to emulsifying wax. Oil-in-watercreams may be formulated using an emulsifying agent such as cetomacrogolemulsifying wax. Suitable properties include the ability to modify theviscosity of the emulsion and both physical and chemical stability overa wide range of pH. The water soluble or miscible cream base may containa preservative system and may also be buffered to maintain an acceptablephysiological pH.

Foam preparations may be formulated to be delivered from a pressurizedaerosol canister, via a suitable applicator, using inert propellants.Suitable excipients for the formulation of the foam base include, butare not limited to, propylene glycol, emulsifying wax, cetyl alcohol,and glyceryl stearate. Potential preservatives include methylparaben andpropylparaben.

Preferably the agents of the invention are combined with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition for use in or on a medical device. Suitablecarriers and diluents include isotonic saline solutions, for examplephosphate-buffered saline. Suitable diluents and excipients alsoinclude, for example, water, saline, dextrose, glycerol, or the like,and combinations thereof. In addition, if desired substances such aswetting or emulsifying agents, stabilizing or ph buffering agents mayalso be present.

The term “pharmaceutically acceptable carrier” refers to anypharmaceutical carrier that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich can be administered without undue toxicity. Suitable carriers canbe large, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, and amino acid copolymers.

Pharmaceutically acceptable salts can also be present, e.g., mineralacid salts such as hydrochlorides, hydrobromides, phosphates, sulfates,and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like.

Suitable carrier materials include any carrier or vehicle commonly usedas a base for creams, lotions, gels, emulsions, lotions or paints fortopical administration. Examples include emulsifying agents, inertcarriers including hydrocarbon bases, emulsifying bases, non-toxicsolvents or water-soluble bases. Particularly suitable examples includepluronics, HPMC, CMC and other cellulose-based ingredients, lanolin,hard paraffin, liquid paraffin, soft yellow paraffin or soft whiteparaffin, white beeswax, yellow beeswax, cetostearyl alcohol, cetylalcohol, dimethicones, emulsifying waxes, isopropyl myristate,microcrystalline wax, oleyl alcohol and stearyl alcohol.

The pharmaceutically acceptable carrier or vehicle may a gel, forexample a nonionic polyoxyethylene-polyoxypropylene copolymer gel, forexample, a Pluronic gel, preferably Pluronic F-127 (BASF Corp.). Thisgel is a liquid at low temperatures but rapidly sets at physiologicaltemperatures, which confines the release of the agent to the site ofapplication or immediately adjacent that site.

An auxiliary agent such as casein, gelatin, albumin, glue, sodiumalginate, carboxymethylcellulose, methylcellulose, hydroxyethylcelluloseor polyvinyl alcohol may also be included in the formulation of theinvention.

Other suitable formulations for coating or imprenating devices includepluronic gel-based formulations, carboxymethylcellulose(CMC)-basedformulations, and hyroxypropylmethylcellulose(HPMC)-based formulations.The composition may be formulated for any desired form of delivery.Other useful formulations include slow or delayed release preparations.

Where the anti-connexin agent is a nucleic acid, such as apolynucleotide, uptake of nucleic acids by mammalian cells is enhancedby several known transfection techniques for example those including theuse of transfection agents. Such techniques may be used with certainanti-connexin agents, including polynucleotides. The formulation whichis used may contain such transfection agents. Examples of these agentsinclude cationic agents (for example calcium phosphate and DEAE-dextran)and lipofectants (for example Lipofectam™ and Transfectam™), andsurfactants.

Where the anti-connexin agent comprises a polynucleotide, theformulation may also includes a surfactant to assist with polynucleotidecell penetration or the formulation may contain any suitable loadingagent. Any suitable non-toxic surfactant may be included, such as DMSO.Alternatively a transdermal penetration agent such as urea may beincluded.

The effective dose for a given device can be determined by routineexperimentation or other methods known in the art or later developed.For example, in order to formulate a range of dosage values, cellculture assays and animal studies can be used. The dosage of suchcompounds preferably lies within the dose that is therapeuticallyeffective for at least 50% of the population, and that exhibits littleor no toxicity at this level.

The effective dosage of each of the anti-connexin agents employed in thedevices of the invention may vary depending on a number of factorsincluding the particular anti-connexin agent or agents employed, thecombinational partner, the mode of administration, the frequency of useof the device or whether it is implanted, the condition being treatedusing the device, etc.

A suitable amount may be from about 0.001 to about 1 mg/kg body weightsuch as about 0.01 to about 0.4 mg/kg body weight. A suitable dose mayhowever be from about 0.001 to about 0.1 mg/kg body weight such as about0.01 to about 0.050 mg/kg body weight. Doses from about 1 to 100,100-200, 200-300, 300-400, and 400-500 micrograms as well as 500-750 and750-1000 micrograms are appropriate.

For example, in certain embodiments, the anti-connexin agent compositionmay be delivered using about 0.01 micromolar (μM) or 0.05 μM to about200 μM final concentration at the treatment site and/or adjacent to thetreatment site. Preferably, the antisense polynucleotide composition isapplied at about 0.05 μM to about 100 μM final concentration, morepreferably, the anti-connexin agent composition is applied at about 1.0μM to about 50 μM final concentration, and more preferably, theanti-connexin agent composition is applied at about 5-10 μM to about30-50 μM final concentration. Additionally, the combined anti-connexinagent composition is applied at about 8 μM to about 20 μM finalconcentration, and alternatively the anti-connexin agent composition isapplied at about 10 μM to about 20 μM final concentration, or at about10 to about 15 μM final concentration. In certain other embodiments, theanti-connexin agent is applied at about 10 μM final concentration. Inyet another embodiment, the anti-connexin agent composition is appliedat about 1-15 μM final concentration. Anti-connexin agent dose amountsinclude, for example, about 0.1-1, 1-2, 2-3, 3-4, or 4-5 micrograms(μg), from about 5 to about 10 μg, from about 10 to about 15 μg, fromabout 15 to about 20 μg, from about 20 to about 30 μg, from about 30 toabout 40 μg, from about 40 to about 50 μg, from about 50 to about 75 μg,from about 75 to about 100 μg, from about 100 μg to about 250 μg, andfrom 250 μg to about 500 μg. Dose amounts from 0.5 to about 1.0milligrams or more or also provided, as noted above. Dose volumes willdepend on the size of the site to be treated, and may range, forexample, from about 25-100 μL to about 100-200 μL, from about 200-500 μLto about 500-1000 μL. Milliliter doses are also appropriate for largertreatment sites.

Still other dosage levels between about 1 nanogram (ng)/kg and about 1mg/kg body weight per day of each of the agents described herein. Incertain embodiments, the dosage of each of the subject compounds willgenerally be in the range of about 1 ng to about 1 microgram per kg bodyweight, about 1 ng to about 0.1 microgram per kg body weight, about 1 ngto about 10 ng per kg body weight, about 10 ng to about 0.1 microgramper kg body weight, about 0.1 microgram to about 1 microgram per kg bodyweight, about 20 ng to about 100 ng per kg body weight, about 0.001 mgto about 100 mg per kg body weight, about 0.01 mg to about 0.1 mg per kgbody weight, or about 0.1 mg to about 1 mg per kg body weight. Incertain embodiments, the dosage of each of the subject compounds willgenerally be in the range of about 0.001 mg to about 0.01 mg per kg bodyweight, about 0.01 mg to about 0.2 mg per kg body weight, about 0.1 mgto about 1 mg per kg body weight. If more than one anti-connexin agentis used, the dosage of each anti-connexin agent need not be in the samerange as the other. For example, the dosage of one anti-connexin agentmay be between about 0.01 mg to about 1 mg per kg body weight, and thedosage of another anti-connexin agent may be between about 0.1 mg toabout 0.5 mg per kg body weight.

Conveniently, the anti-connexin agent is administered in a sufficientamount to downregulate or inhibit expression of a connexin protein, ormodulate gap junction formation or connexon opening for at least about0.5 to 1 hour, at least about 1-2 hours, at least about 2-4 hours, atleast about 4-6 hours, at least about 6-8 hours, at least about 8-10hours, at least about 12 hours, or at least about 24 hourspost-administration.

The dosage of each of the anti-connexin agents in the compositions andmethods of the subject invention may also be determined by reference tothe concentration of the composition relative to the size, length,depth, area or volume of the area to which it will be applied. Forexample, in certain dosing of the pharmaceutical compositions may becalculated based on mass (e.g. micrograms) of or the concentration in apharmaceutical composition (e.g. μg/μl) per length, depth, area, orvolume of the area of application.

Agents useful for wound healing suitable for the preparation of thepharmaceutical compositions described herein may be prepared andadministered using methods as known in the art (see, for example, U.S.Pat. Nos. 7,098,190, 6,319,907, 6,331,298, 6,387,364, 6,455,569,6,566,339, 6,696,433, 6,855,505, 6,900,181, 7,052,684 and EP1100529 B1.The concentration of each anti-connexin agent and agents useful forwound healing need not be in the same range as the other. Other amountswill be known to those of skill in the art and readily determined. Forexample, suitable combination dosages and formulations in accordancewith various aspects and embodiments as described herein may beadministered according to the dosing regimen as described in U.S. Pat.No. 6,903,078 to Lewis entitled “Combination PDGF, KGF, IGF, and IGFBPfor wound healing.”

The wound healing agent may be used in conjuction with a medical devicedirected towards any tissue. An effective dose of PDGF has been reportedto be 5 ng/mm² or higher when applied topically as described in U.S.Pat. No. 4,861,757, and at least 1 ng/ml local concentration of anisoform of PDGF (for example, PDGF-AA, PDGF-BB, or PDGF-AB), up to about30 ng/ml local concentration applied to a population of fibroblasts asdescribed in Lepisto et al., Biochem Biophys Res. Comm 209: 393-399(1995). PDGF can be administered in a carboxymethylcellulose gelformulation at concentrations of about 10 μg/gm to about 500 μg/gm ofgel, about 20 μg/gm to about 200 μg/gm, and about 30 μg/gm to about 100μg/gm of gel, optimally about 100 μg/gm of gel. Efficacy of PDGF hasbeen achieved within the range of about 3 μg/ml solution to about 300μg/ml of solution administered.

About 50 μl of KGF of a concentration of about 5 μg/ml may be effectivefor wound healing by topical application to epithelial tissue asdescribed in Sotozono et al, Invest. Opthal. Vis. Science 36: 1524-29(1995). As described in U.S. Pat. No. 4,861,757, an effective amount ofIGF when co-administered with PDGF is in the range of at least 2.5ng/mm² to about 5 ng/mm², with a ratio of PDGF to IGF in the range ofabout 1:10 to about 25:1 weight to weight, with the most effectiveratios being PDGF to IGF of about 1:1 to about 2:1 weight to weight.IGFBP administered in combination with IGF has been shown to increasewound healing at dose levels of about 5 μg of IGF with about 1.5 μg ofphosphorylated IGFBP in a molar ration of about 11:1 IGF:IGFBP, asdescribed in Jyung et al, Surgery 115:233-239 (1994).

For administration of polypeptide therapeutics, for example, PDGF, KGF,IGF and IGFBP polypeptides, the dosage can be in the range of about 5 μgto about 50 μg/kg of tissue to which the application is directed, alsoabout 50 μg to about 5 mg/kg, also about 100 μg to about 500 μg/kg oftissue, and about 200 to about 250 μg/kg. For polynucleotidetherapeutics, for example in a gene therapy administration protocol,depending on the expression strength the polynucleotide in the patient,for tissue targeted administration, vectors containing expressibleconstructs including PDGF, KGF, IGF, and IGFBP coding sequences can beadministered in a range of about 100 ng to about 200 mg of DNA for localadministration in a gene therapy protocol, also about 500 ng to about 50mg, also about 1 μg to about 2 mg of DNA, about 5 μg of DNA to about 500μg of DNA, and about 20 μg to about 100 μg during a local administrationin a gene therapy protocol, and about 250 μg, per injection oradministration. Factors such as method of action and efficacy oftransformation and expression are therefore considerations that willeffect the dosage required for ultimate efficacy for administration ofDNA therapeutics. Where greater expression is desired, over a largerarea of tissue, larger amounts of DNA or the same amountsre-administered in a successive protocol of administrations, or severaladministrations to different adjacent or close tissue portions of forexample, a wound site may be required to effect a positive therapeuticoutcome.

Therapeutic agents and gap junction modifying agents suitable for thepreparation of the medical devices described herein may be formulatedusing methods as known in the art.

As noted herein, the doses of either an anti-connexin agent or anotheragent administered in combination can be adjusted down from the dosesadministered when given alone.

The combined use of several agents may reduce the required dosage forany individual agent because the onset and duration of effect of thedifferent agents may be complementary. In a preferred embodiment, thecombined use of one or more anti-connexin agents and one or moretherapeutic agents, agents useful for wound healing, and/or gap junctionmodifying agents has an additive, synergistic or super-additive effect.

In some cases, the combination of one or more anti-connexin agents andone or more therapeutic agents and/or one or more agents useful forwound healing, and/or one or more gap junction modifying agents have anadditive effect. In other cases, the combination can havegreater-than-additive effect. Such an effect is referred to herein as a“supra-additive” effect, and may be due to synergistic or potentiatedinteraction.

The term “supra-additive promotion of wound healing” refers to a meanwound healing produced by administration of a combination of ananti-connexin agent and one or more therapeutic agents; agents usefulfor wound healing and/or gap junction modifying agents, is statisticallysignificantly higher than the sum of the wound healing produced by theindividual administration of either any of the agents alone. Whetherproduced by combination administration of an anti-connexin agent and oneor more therapeutic agents, agents useful for wound healing and/or gapjunction modifying agents is “statistically significantly higher” thanthe expected additive value of the individual compounds may bedetermined by a variety of statistical methods as described hereinand/or known by one of ordinary skill in the art. The term “synergistic”refers to a type of supra-additive inhibition in which both theanti-connexin agent and one or more therapeutic agents, agents usefulfor wound healing and/or gap junction modifying agents individually havethe ability to promote wound healing or reduce fibrosis and scarring.The term “potentiated” refers to type of supra-additive effect in whichone of the anti-connexin agent or one or more therapeutic agents, agentsuseful for wound healing and/or gap junction modifying agentsindividually has the increased ability to promote wound healing.

In general, potentiation may be assessed by determining whether thecombination treatment produces a mean wound healing increase in atreatment group that is statistically significantly supra-additive whencompared to the sum of the mean wound healing increases produced by theindividual treatments in their treatment groups respectively. The meanwound healing increase may be calculated as the difference betweencontrol group and treatment group mean wound healing. The fractionalincrease in wound healing, “fraction affected” (Fa), may be calculatedby dividing the treatment group mean wound healing increase by controlgroup mean wound healing. Testing for statistically significantpotentiation requires the calculation of Fa for each treatment group.The expected additive Fa for a combination treatment may be taken to bethe sum of mean Fas from groups receiving either element of thecombination. The Two-Tailed One-Sample T-Test, for example, may be usedto evaluate how likely it is that the result obtained by the experimentis due to chance alone, as measured by thep-value. Ap-value of less than0.05 is considered statistically significant, that is, not likely to bedue to chance alone. Thus, Fa for the combination treatment group mustbe statistically significantly higher than the expected additive Fa forthe single element treatment, groups to deem the combination asresulting in a potentiated supra-additive effect.

Whether a synergistic effect results from a combination treatment may beevaluated by the median-effect/combination-index isobologram method(Chou, T., and Talalay, P. (1984) Ad. Enzyme Reg. 22:27-55). In thismethod, combination index (CI) values are calculated for differentdose-effect levels based on parameters derived from median-effect plotsof the anti-connexin agent alone, the one or more agents useful forwound healing alone, and the combination of the two at fixed molarratios. CI values of & lt; 1 indicate synergy, CI-1 indicates anadditive effect, and CP1 indicates an antagonistic effect. This analysismay be performed using computer software tools, such as CalcuSyn,Windows Software for Dose Effect Analysis (Biosoft(D, Cambridge UK).

Any method known or later developed in the art for analyzing whether asupra-additive effect exists for a combination therapy is contemplatedfor use in screening for suitable anti-connexin agents for use incombination with one or more therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents.

In another preferred embodiment, the combined use of one or moreanti-connexin agents and one or more therapeutic agents, agents usefulfor wound healing, and/or gap junction modifying agents reduces theeffective dose of any such agent compared to the effective dose whensaid agent administered alone. In certain embodiments, the effectivedose of the agent when used in combination with one or moreanti-connexin agents is about 1/15 to about ½, about 1/10 to about ⅓,about ⅛ to about ⅙, about ⅕, about ¼, about ⅓ or about ½ the dose of theagent when used alone.

In another preferred embodiment, the combined use of one or moreanti-connexin agents and one or more therapeutic agents, agents usefulfor wound healing, and/or gap junction modifying agents reduces thefrequency in which said agent is administered compared to the frequencywhen said agent is administered alone. Thus, these combinations allowthe use of lower and/or fewer doses of each agent than previouslyrequired to achieve desired therapeutic goals.

In one aspect of the invention the anti-connexin agent is administeredin one composition and the therapeutic agent, wound healing agent and/orgap junction modifying agent is administered in a second composition. Inone embodiment the first composition comprising one or moreanti-connexin agents is administered before the second compositioncomprising one or more therapeutic agents, agents useful for woundhealing and/or gap junction modifying agents. In one embodiment thefirst composition comprising one or more anti-connexin agents isadministered after the second composition comprising one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents. In one embodiment the first composition comprising oneor more anti-connexin agents is administered via the medical devicebefore and after the second composition comprising one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents. In one embodiment the first composition comprising oneor more anti-connexin agents is administered via the medical deviceabout the same time as the second composition comprising one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents.

Preferably one or more anti-connexin agents and one or more therapeuticagents, agents useful for wound healing and/or gap junction modifyingagents are delivered by topical application via the medical device,including but not limited to application using solid supports (such asmatrices) and medicinal layers or coatings as described herein orotherwise known in the art. In one embodiment, the solid supportcomprises a biocompatible membrane. In another embodiment, the solidsupport comprises a matrix. In one embodiment of the invention, thesolid support composition may be a slow release solid supportcomposition, in which the one or more anti-connexin agents and one ormore therapeutic agents, agents useful for wound healing and/or gapjunction modifying agents is dispersed in a slow release solid matrixsuch as a matrix of alginate, collagen, or a synthetic bioabsorbablepolymer. Preferably, the solid support composition is sterile or lowbio-burden.

The delivery of one or more anti-connexin agents and one or moretherapeutic agents, agents useful for wound healing and/or gap junctionmodifying agents of the formulation over a period of time via themedical device, in some instances for about 1-2 hours, about 2-4 hours,about 4-6 hours, about 6-8, or about 24 hours or longer, may be aparticular advantage in implanted devices. In some instances, cell lossmay extend well beyond the site of a procedure to surrounding cells.Such loss may occur within 24 hours of the original procedure and ismediated by gap junction cell-cell communication. Administration ofanti-connexin agent(s), e.g., for downregulation or inhibition ofconnexin expression, or blockade of connexon opening, therefore willmodulate communication between the cells, or loss into the extracellularspace in the case of connexon regulation, and minimize additional cellloss or injury or consequences of injury.

While the delivery period will be dependent upon both the site at whichthe downregulation is to be induced and the therapeutic effect which isdesired, continuous or slow-release delivery via the medical device forabout 1-2 hours, about 2-4 hours, about 4-6 hours, about 6-8, or about24 hours or longer is provided. In accordance with the presentinvention, this is achieved by inclusion of the anti-connexin agentsand/or one or more therapeutic agents, agents useful for wound healingand/or gap junction modifying agents in a medical device together with apharmaceutically acceptable carrier, coating, layer or other vehicle,particularly in a form for continuous or slow-release administration.

Various aspects of the invention will now be described with reference tothe following experimental section which will be understood to beprovided by way of illustration only and not to constitute a limitationon the scope of the invention.

EXAMPLES Example 1 Determination of Effects from Use of an Anti-ConnexinAgent-Treated Device in an Animal Model

The following experiment is directed to coating artificial surfaces withan anti-connexin agent for amelioration of tissue damage and/or enhancedtissue repair and their testing in an animal model.

Materials: Sodium bicarbonate, sodium chloride, sodium phosphate, sodiumnitrite, potassium phosphate-monobasic, 40% formaldehyde solution andsucrose are available from Fischer Scientific, Fairlawn, N.J. SephadexG25 is available from Pharmacia, Piscataway, N.J. Monoclonal mouseanti-proliferating cell nuclear antigen is available from Dako A/S,Denmark. All other chemicals are available from Sigma Chemical Co., St.Louis, Mo.

Tris-buffered saline consisted of 10 mM tris[hydroxymethyl]aminoethane,pH 7.4, and 150 mM NaCl. Phosphate-buffered saline contained 10 mMsodium phosphate and 150 mM NaCl, pH 7.4.

Anti-Connexin Agent Species: Anti-connexin agent-BSA is synthesized asfollows: Fatty acid-free bovine serum albumin (200 mg/ml) is exposed toa 1.4 molar-fold excess of anti-connexin agent in PBS for 30 minutes atroom temperature.

Thiolated bovine serum albumin (pS-BSA) is prepared after Benesch andBenesch (Benesch R & Benesch R E, “Preparation and properties ofhemoglobin modified with derivatives of pyridoxal”. Methods Enzymol.,76:147-159 (1981)). Briefly, essential fatty acid-free bovine serumalbumin (50 mg/ml) is dissolved in water with N-acetyl-homocysteinethiolactone (35 mM) and 0.05% polyethylenesorbitan monolaurate.Equimolar Silver nitrate is slowly added at room temperature over 90minutes at pH 8.5. Excess thiourea (70 mM) is added and the pH loweredto 2.5. Excess silver nitrate is removed by Dowex 50 chromatography withthe mobile phase consisting of 1M thiourea, pH 2.5, and excess thioureais removed by Sephadex G-25 chromatography. The pS-BSA is preparedwithin two days of subsequent derivitization and stored at 4° C.Derivitization of pS-BSA is accomplished with 1.4 fold molar excess5-triethylenetetramine in 0.5 N HCl for 30 minutes at room temperature.The solution is adjusted to pH 4.0 with 0.5 N NACH after derivitization.Protein content is determined using the method of Lowry and colleagues(Marcus Salier, FASEB. J., 7:516-522, 1993).

Animal Preparation: All animal preparations are performed withininstitutional guidelines of the host institution. New Zealand whiterabbits (3.5-4.2 kg) of either sex are premedicated with 5 mg/kgintramuscular (IM) xylazine hydrochlolide (Miles Pharmaceuticals,Shawnee Mission, Kans.), and 0.1 mg/kg subcutaneous (SC) atropinesulfate (Lyphomed, Deerfield, Ill.) fifteen minutes prior to theinduction of anesthesia. Anaesthesia is induced with 40 mg/kg IMketamine hydrochloride (Fort Dodge Laboratories, Fort Dodge, Iowa) and 5mg/kg IM acepromazine maleate (Aveco Company, Inc., Fort Dodge, Iowa).Additional doses of ketamine hydrochloride are administered as necessaryto maintain anesthesia. For survival studies, 100,000 U penicillin G(Apothecon of Bristol-Myers Squibb, Princeton, N.J.), is administered IMperioperatively. The skin over the femoral arteries is infiltrated with1% lidocaine (Astra Pharmaceuticals, Inc., Westborough, Mass.), and thecommon femoral arteries are exposed from the inguinal ligament to thesuperficial femoral artery. Arteries are cleared of connective tissue,side branches are ligated, and the superficial femoral artery issuspended with silk ties. A 1.5-to-2.0 cm length of femoral artery isisolated from the circulation proximally and distally with neurosurgicalmicroaneurysm clips. The superficial femoral artery is cannulated with aS-triethylenetetramine-BSA coated 2F Fogarty balloon catheter (AmericanEdwards Laboratories, Santa Ana, Calif.), that is passed into theisolated segment of femoral artery. The balloon is inflated withsufficient air to generate slight resistance and withdrawn three times.The contralateral femoral artery is prepared identically as anappropriate control, i.e., using a balloon catheter coated withunderivatized BSA. Following removal of the balloon catheter, thesuperficial femoral artery is ligated and flow re-established. The areaof balloon injury is marked by surgical staples in the adjacent musclefascia. The incision is closed with subcuticular absorbable suture andthe animals allowed to recover. In some experiments, a distant controlvessel, the right carotid artery, is isolated and harvested without anyother manipulation.

Tissue Processing and Analysis: On the 14th postoperative day, animalsare euthanized with 120 mg/kg intravenous sodium pentobarbital (AnproPharmaceuticals, Arcadia, Calif.), and the abdominal aorta and inferiorvena cava interrupted by silk ties. A 7F plastic cannula is insertedinto the abdominal aorta and the vessels perfused clear with salinefollowed by fixation at 100 mm Hg pressure with 10% buffered formalin.The vessels are stored in 10% buffered formalin and the samplesparaffin-embedded and microtome-sectioned. Six sections are made alongthe length of each injured segment of vessel and stained with Verhoeff'sstain for elastic tissue. The areas within the lumen, internal elasticmembrane, and external elastic membrane are measured by a blindedobserver using computerized digital planimetry (Zeiss, West Germany).The areas within the lumen, internal elastic membrane and externalelastic membrane are analyzed. Sections with obstructive thrombusimpairing analysis are discarded.

In a separate set of animals, vessels are perfusion-fixed with 10%buffered formalin seven days after injury and processed for analysis ofproliferating cells within 12 hours. Sections are stained forproliferating cell nuclear antigen (PCNA) and adjacent sections arestained with hematoxylin and eosin. Five representative sections fromeach segment are examined. Total nuclei are counted from the hematoxylinand eosin slides and percent PCNA positive cells are defined as thenumber of PCNA-positive nuclei divided by the total number of nucleimultiplied by 100.

Statistics: Treatments are administered in a paired fashion with onefemoral artery cannulated with an anti-connexin agent-BSA coated ballooncatheter while the other femoral artery is cannulated with anunderivatized BSA coated balloon catheter. Data is tested for normalityusing appropriate statistical methods, for example, theKolmogorov-Smirnov algorithm and for equal variance with the LeveneMedian test. Normally distributed variables are compared using, forexample, the paired t-test and non-normally distributed variables using,for example, the Wilcoxon sign-ranks test or the Mann-Whitney rank-sumtest. Non-paired data are compared using, for example, an independentt-test. Statistical significance is accepted if the null hypothesis isrejected with P<0.05.

Anti-Connexin Agent-BSA Effect on Platelet Binding to Injured Vessel:Platelet adhesion to the injured arterial surface has been reported tobe important in the proliferative response to injury. Accordingly theeffects of anti-connexin agent-BSA on platelet deposition after ballooninjury are investigated. Platelet deposition is assessed at the site ofcannulation with anti-connexin agent-BSA coated balloon catheter, andcompared to that at the site of cannulation with an underivatized BSAcoated balloon catheter. Decreased platelet deposition at the site ofcannulation with anti-connexin agent-BSA coated balloon catheter isindicative of ameliorated tissue damage and/or enhanced tissue repair.

Anti-connexin agent-BSA Effects on Neointimal Proliferation: Neointimalproliferation after local delivery of anti-connexin agent-BSA andappropriate controls are evaluated by comparing absolute neointimal areaand neointima/media ratios. The absolute neointimal area andneointima/media ratio at the site of cannulation with an anti-connexinagent-BSA coated balloon catheter is assessed and compared to aneointimal area and neointima/media ratio at the site of cannulationwith an underivatized BSA coated balloon catheter. A decrease inneointimal area or reduction in the neointima/media ratio is indicativeof an inhibition of neointimal proliferation and of amelioration oftissue damage and/or enhanced tissue repair.

Anti-connexin agent-BSA Effects on Cellular Proliferation: Mousemonoclonal antibody staining against PCNA is used to assay the degree ofS1-phase activity at 7 days after injury. At this time, the percent ofproliferating cells is assessed in vessels cannulated with ananti-connexin agent-BSA coated balloon catheter and in vesselscannulated with an underivatized BSA coated balloon catheter.Histological assessment determines the cellular populations undergoingproliferation.

These experiments are directed to the effect on neointimal proliferationand amelioration of tissue damage and/or enhanced tissue repair bylocalised delivery of an anti-connexin agent by, for example, thecoating of a balloon catheter with anti-connexin agent-BSA.

The endothelium is reported to be essential for vascular integrity,control of thrombosis, (Clowes et al., Lab. Invest. 49:327-333, 1983);(Rees et al., Proc. Natl. Acad. Sci. USA. 86:3375-3378, 1989) and theregulation of intimal growth (Kubes et al., Proc. Natl. Acad. Sci. USA,88:4651-4655, 1991), and has been proposed to be important in the localcontrol of vascular smooth muscle growth. Balloon angioplasty reportedlyremoves the endothelium from arterial smooth muscle, and theseendothelial functions can often be lost during the procedure. Inparticular, removal of the endothelium and damage to the smooth musclecells have been reported to be associated with intimal proliferation(McNamara et al., Biochem. Biophys. Res. Commun., 193:291-296, 1993).The mechanism for this response is complex and reportedly involvesplatelet deposition and activation, cytokine elaboration, smooth musclecell migration and proliferation, and extra-cellular matrix production.It has been reported that after balloon injury, the endotheliumregenerates rapidly but is often dysfunctional, (Saville, Analyst83:670-672, 1958).

A limitation of neointimal proliferation after a single, localadministration of an anti-connexin agent is indicative of ameliorationof tissue damage and/or enhanced tissue repair. Anti-platelet activitymay explain such findings. Inhibition of platelet binding has been saidto result in many effects that are likely to reduce the proliferativeresponse after injury. For example, platelet adhesion and aggregation issaid to be associated with the release of PDGF, basic fibroblast growthfactor, epidermal growth factor, and transforming growth factor-β,potent stimuli for smooth muscle cell proliferation and matrixproduction.

There may additionally or alternatively be a direct effect on vascularsmooth muscle gene expression, migration, proliferation or synthesis ofextracellular matrix. Such an effect may be in addition to anamelioration of tissue damage and/or enhancement in tissue repair by,for example, restoration of normal tissue stem cell responses.

It has been reported that the mechanical removal of the endotheliumabolishes the vasodilator responses to endothelium-dependent vasoactivestimuli, while leaving the vasoconstrictor effects of agonists to smoothmuscle unopposed (Furchgott Zawadzki, Nature 288:373-376 (1980). Thisprocess reportedly occurs with balloon angioplasty especially at siteswhere platelet thrombus is noted (Uchida et al., Am. Heart. J.,117:769-776 (1989); Steele et al., Circ. Res., 57:105-112 (1985). Thestrategy of administration of an anti-connexin agent as therapy for, forexample, acute thrombotic phenomena and restenosis following angioplastyis supported by results indicating an amelioration of tissue damageand/or enhancement in tissue repair.

Example 2 Preparation and Use of an Anti-connexin Agent Coated MedicalDevice for Ameliorating Tissue Damage and/or Enhancing Tissue Repair

The following experiment is directed to application of anti-connexinagents to coated artificial surfaces, such as, for example, syntheticvascular graft material, or stents, for use in ameliorating tissuedamage and/or enhancing tissue repair.

First, dacron grafts and cardiac catheters are coated with afunctionalized anti-connexin agent, such as for example, anti-connexinagent-bovine serum albumin (BSA). In three separate experiments, anidentical pair of 6 mm (internal diameter) knitted dacron grafts, 5 cmin length, are prepared for surgical placement in the transected carotidarteries of six anesthetized dogs. For each experiment, three controldogs receive grafts coated in underivatized BSA, while for each of thethree trial dogs, one graft is soaked in 5% BSA and the other graft issoaked in 5% BSA combined with 0.5 mM anti-connexin agent producing,anti-connexin agent-BSA, an example of a functionalized anti-connexinagent protein, for one hour prior to insertion, and then rinsed insaline. The grafts are sutured in place with a continuous 6-0 prolinesuture.

Following graft insertion, the dogs are observed for two months.

Histological examination of the site of insertion is performed at theend of the two month period. Amelioration of tissue damage or enhancedtissue repair is evidenced by, for example, the appearance of normalvascular epithelial cells at the site of graft insertion. Tissue repairat the site of anti-connexin agent-BSA-coated graft insertion iscompared to that at the site of underivatized, BSA-coated graftinsertion.

Evidence of amelioration of tissue damage and/or enhanced tissue repairat the site of anti-connexin agent-BSA coated graft insertion comparedto that at the site of underivatized BSA-coated graft insertion is shownby amelioration of tissue damage and/or enhanced tissue repair at thesite of insertion of the synthetic grafts treated with anti-connexinagent-BSA during exposure of the graft to circulating blood over aperiod of two months.

Example 3 Application of Anti-Connexin Agents to Damaged VascularSurfaces to Ameliorate Tissue Damage and/or Enhance Tissue Repair

The following experiment is directed to application of anti-connexinagents to damaged vascular surfaces, for example, arterial surfaces, toameliorate tissue damage and/or enhance tissue repair.

In five anesthetized dogs, both carotid arteries are exposed. Two 3 FRUSC1 catheters are prepared for arterial implantation. One catheter issoaked in a 5% BSA solution for 12 hours, while the other is soaked in a5% BSA solution which also contains 1 mg/ml of test anti-connexin agent.One each of the two coated catheters is placed randomly in the right orleft carotid artery of the dog through a small incision sealed with a6-0 proline suture. The catheters are advanced for 5 cm into thearterial lumen. Following catheter insertion, the dogs are observed fortwo weeks.

Histological examination of the site of insertion is performed at theend of the two week period. Lessened tissue damage, and/or tissuerepair, is evidenced by, for example, the appearance of normal vascularepithelial cells at the site of catheter insertion. Tissue at the siteof anti-connexin agent-BSA coated catheter insertion is compared to thatat the site of underivatized BSA-coated catheter insertion.

Evidence of enhanced tissue repair and/or amelioration of tissue damageat the site of anti-connexin-BSA coated catheter insertion compared tothat at the site of underivatized BSA-coated catheter insertion is shownby enhanced tissue repair and/or amelioration of tissue damage at thesite of insertion of the catheter treated with anti-connexin agent-BSAduring exposure of the catheter to circulating blood over a period oftwo weeks.

Example 4 Determination of Tissue Damage Using an Anti-Connexin AgentTreated Medical Device in a Pig Model

The following experiment is directed to determining the tissue damageusing a treated medical device in a pig model.

Pigs are subjected to coronary balloon-injury using standard methods.Prior to balloon injury, an angiogram is performed. Thereafter, in trialpigs, a test anti-connexin agent-BSA coated balloon catheter, forexample, is used for balloon injury, whereas in control pigs, anunderivatized BSA coated balloon catheter is used. The balloon of thecatheter is inflated for 15 min, then deflated and the catheter isremoved. Another angiogram is performed 30 minutes after injury todetermine the degree of spasm. Coronary catheters are placed in thecoronary ostea, radiocontrast is infused into the coronary arteries andmeasurements are made of the degree of so-called “recoil spasm” thatexists at the point of angioplasty. The degree of spasm or recoil isdefined quantitatively, again using the computer-driven quantitativecoronary angiography algorithm that compares the segment at the site ofballoon injury with a proximal segment that is uninjured as a referencestandard. All catheters are then removed and incision sites repaired.The animals are awakened and maintained with normal chow diets over thenext four weeks. At the end of that period of time, the animals areagain sedated, undergo coronary angiography to determine coronarystenoses at the site of angioplasty. Catheters are placed in thecoronary ostea and radiocontrast fluid is infused. The angiograms arerecorded and subsequently processed by a computer-driven quantitativecoronary angiography algorithm to determine lumen diameter. The degreeof stenosis represents the percentage reduction in the lumen diametercompared with a reference segment proximal to the area of stenosis usingstandard methods. The animals are euthanized by an overdose ofpentobarbital. Their coronary arteries are perfusion fixed with formalinat 100 mm Hg of perfusion pressure, harvested and sectioned forquantitative morphometric assessment of the lumen diameter, theneointimal dimension and cross-section, as well as the neointimal area.The arteries are stained with hematoxylin and eosin. The neointima tolumen diameter ratio is determined and is compared, between trial andcontrol animals.

Example 5 Determination of Effect on Tissue Damage and/or Tissue RepairUsing an Anti-Connexin Agent Coated Medical Device

This experiment is directed to coating a Palmaz-Schatz stent with ananti-connexin agent to reduce the degree and severity of neointimalhyperplasia leading to restenosis. Palmaz-Schatz stents are dip-coatedin 800-1000 μM test anti-connexin agent-BSA, for example, orunderivatized BSA three times for 10 minutes followed by 10 minutes ofair drying time. One test anti-connexin agent-BSA coated and oneunderivatized BSA-coated stent is placed under sterile conditions in thecarotid arteries of 10 pigs, one in each carotid artery. They arefollowed for 28 days and then the carotid arteries are removed. They areexamined histologically for the degree of neointimal hyperplasia, andtissue damage/repair. Tissue repair is evidenced by, for example, theappearance of normal vascular epithelial cells at the site of insertionof the stent. Tissue damage/repair at the site of test anti-connexinagent-BSA coated stent insertion is compared to that at the site ofunderivatized BSA-coated stent insertion.

Evidence of amelioration of tissue damage and/or enhanced tissue repairat the site of test anti-connexin agent-BSA coated stent insertioncompared to that at the site of underivatized BSA-coated stent insertionshows that during exposure of the stent to circulating blood, there isamelioration of tissue damage and/or enhanced tissue repair at the siteof insertion of the stent coated with anti-connexin agent, and thatdelivery of an anti-connexin agent is able to ameliorate tissue damageenhance tissue repair.

Example 6 Method of Coating Pharmaceutical Agents onto Sutures

A 4-0 VICRYL (Polyglactin 910) Suture (Ethicon, Somerville, N.J.) iscoated with suitable anti-connexin agent and gelatin. The coatingsolution comprises of 4 ml gelatin solution and 2 ml of anti-connexinagent solution. The gelatin component is prepared by heating a 10 wt %solution of medical grade soluble bovine collagen (Semed-S, Kensey-Nash,Exton, Pa.) to 80° C. for 10 minutes followed by incubation at 37° C.The anti-connexin agent, e.g., an anti-connexin 43 agent (for example, aconnexin 43 antisense ODN, or an anti-connexin 43 peptide orpeptidomimetic, such as one that blocks or inhibits hemichannelopening), is prepared at suitable concentrations, such as, for example,10, 3, 0.6, and 0 μg/ml. The resulting concentrations in the coatingsolutions can then be determined. The coating solutions are kept at 37°C. until use. Prior to coating, the sutures are pretreated with a bathof 70% ethanol solution for 10 minutes, followed by a wash with saline.The suture is then placed in the coating solution and incubated at 37°C. for 30 minutes with gentle agitation. The suture is then removed fromthe solution and is then air-dried overnight.

The concentration of the anti-connexin agent on the suture is quantifiedby an ELISA method. The anti-connexin agent is first eluted from a 4 cmsegment of suture in 2 ml of 6M Urea solution (75 mM NaH2PO 4, pH 2.7)at 37° C. for 1 hour. The elution solutions are analyzed by a sandwichELISA assay that detects the presence of the anti-connexin agent. Theconcentration of the anti-connexin agent on the suture is measured inμg/cm.

Example 7

A 0 ETHIBOND EXCEL Polyester Suture (Ethicon, Somerville, N.J.) iscoated with anti-connexin agent and gelatin in a similar manner asdescribed in Example 6. An anti-connexin agent solution is concentratedto appropriate concentration (in mg/ml) with a centrifugal filter device(e.g. Centriplus YM-10, Regenerated Cellulose 10,000 MWCO, AmiconBioseparations). The coating solution comprises of 0.5 ml concentratedanti-connexin solution and 1 ml 10 wt % gelatin solution. Theconcentration of anti-connexin solution on the coated suture, asquantified by ELISA, is then measured.

Sutures are pulled through a sample of goat ACL tissue to evaluate ifany of the anti-connexin agent coating is sheared off during its use.The concentration of anti-connexin agent post-surgery is measured. Ifappropriate concentration is obtained, it is an indication that thegelatin is effective in maintaining the anti-connexin agent on thesuture even while passing through tissue.

Preferred anti-connexin agents for sutures areanti-connexin 43 agent,particularly connexin 43 antisense (e.g., ODN) molecules, or ananti-connexin 43 peptide or peptidomimetic, such as one that blocks orinhibits hemichannel opening), or both together,

Example 8

A 0 Plain Surgical Gut Suture (Ethicon, Somerville, N.J.) is coated withanti-connexin agent. The coating solution comprises of 1 mlanti-connexin agent solution concentrated to the appropriateconcentration (in mg/ml) with a centrifugal filter device (e.g.Centriplus YM-10, Regenerated Cellulose 10,000 MWCO, AmiconBioseparations). The gut suture is pretreated in a bath of 200 mM NaH2PO4 (pH 11.2) for 10 minutes followed by a wash in PBS prior tocoating. The concentration of the anti-connexin coating on the coatedgut suture is then quantified by ELISA assay.

Example 9

A partially resorbable composite suture (e.g., 2 ORTHOCORD OrthopaedicSuture, DePuy Mitek, Raynham, Mass.) is coated with anti-connexin agentand gelatin in a similar manner as described in Example 6. The coatingsolution comprises of suitable volume of anti-connexin agent solution ata desired concentration (e.g. 3.5 mg/ml and 1.4 ml 10 wt % gelatinsolution). The concentration of the anti-connexin agent on the coatedpartially resorbable composite suture is then quantified by ELISA (e.g.μg/cm).

All patents, publications, scientific articles, web sites, and otherdocuments and materials referenced or mentioned herein are indicative ofthe level of skill of those skilled in the art to which the inventionpertains, and each such referenced document and material is herebyincorporated by reference to the same extent as if it had beenincorporated by reference in its entirety individually or set forthherein in its entirety. Applicants reserve the right to physicallyincorporate into this specification any and all materials andinformation from any such patents, publications, scientific articles,web sites, electronically available information, and other referencedmaterials or documents.

The written description portion of this patent includes all claims.Furthermore, all claims, including all original claims as well as allclaims from any and all priority documents, are hereby incorporated byreference in their entirety into the written description portion of thespecification, and Applicants reserve the right to physicallyincorporate into the written description or any other portion of theapplication, any and all such claims. Thus, for example, under nocircumstances may the patent be interpreted as allegedly not providing awritten description for a claim on the assertion that the precisewording of the claim is not set forth in haec verba in writtendescription portion of the patent.

The claims will be interpreted according to law. However, andnotwithstanding the alleged or perceived ease or difficulty ofinterpreting any claim or portion thereof, under no circumstances mayany adjustment or amendment of a claim or any portion thereof duringprosecution of the application or applications leading to this patent beinterpreted as having forfeited any right to any and all equivalentsthereof that do not form a part of the prior art.

All of the features disclosed in this specification may be combined inany combination. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Thus,from the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention. Other aspects, advantages,and modifications are within the scope of the following claims and thepresent invention is not limited except as by the appended claims.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,the terms “comprising”, “including”, “containing”, etc. are to be readexpansively and without limitation. In particular, the phrase “forexample” shall be interpreted to mean “for example, and including butnot limited to.” The methods and processes illustratively describedherein suitably may be practiced in differing orders of steps, and thatthey are not necessarily restricted to the orders of steps indicatedherein or in the claims.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by various embodiments and/or preferredembodiments and optional features, any and all modifications andvariations of the concepts herein disclosed that may be resorted to bythose skilled in the art are considered to be within the scope of thisinvention as defined by the appended claims.

Other embodiments are within the following claims. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically and/orexpressly disclosed herein. Under no circumstances may the patent beinterpreted to be limited by any statement made by any Examiner or anyother official or employee of the Patent and Trademark Office unlesssuch statement is specifically and without qualification or reservationexpressly adopted in a responsive writing by Applicants.

1. A medical device for use or implantation in a mammal, comprising ananti-connexin 43 compound releasable upon insertion of the medicaldevice to or within a subject.
 2. The medical device of claim 1, whereinthe anti-connexin 43 compound decreases connexin 43 protein expression.3. The medical device of claim 1 wherein the anti-connexin 43 compoundis an antisense oligonucleotide.
 4. The medical device of claim 1wherein the anti-connexin 43 compound is an siRNA or RNAioligonucleotide.
 5. The medical device of claim 1 wherein theanti-connexin 43 compound is a peptide compound.
 6. The medical deviceof claim 5 wherein the peptide compound is a peptidomimetic.
 7. Themedical device of claim 1, wherein the anti-connexin 43 compound is ananti-connexin 43 antibody, F(v) fragment, Fab, fragment, Fab′ fragmentor F(ab′)₂ fragment.
 8. The medical device of claim 9, wherein theantibody or antibody fragment is chimeric or humanized antibody.
 9. Themedical device of claim 1, wherein the anti-connexin 43 agent binds toconnexin 43 mRNA.
 10. The medical device of claim 1, wherein theanti-connexin agent binds to a connexin 43 hemichannel.
 11. The medicaldevice of claim 10, wherein the anti-connexin agent binds to ahemichannel extracellular loop.
 12. The medical device of claim 1,wherein the anti-connexin agent is in a polynucleotide having a sequenceselected for SEQ.ID.NOS.1 or 2 or a pharmaceutically acceptable saltthereof.
 13. The medical device of claim 1, wherein the anti-connexinagent is a peptide having a sequence selected from SEQ. ID.NOS. 15 to 38or a pharmaceutically acceptable salt thereof.
 14. The medical device ofclaim 1, wherein the anti-connexin agent is a peptide of SEQ.ID.NOS. 18or
 19. 15. The medical device of claim 1, wherein the anti-connexinagent is a peptide of SEQ.ID.NO.19.
 16. The medical device of claim 1,wherein the anti-connexin agent is a polynucleotide having a sequenceselected from the group consisting of: SEQ.ID.NOS.1 to
 12. 17. Themedical device of claim 1, wherein the surface of the medical devicecomprises an anti-connexin agent.
 18. The medical device of claim 17,wherein the anti-connexin agent is present at a weight percentage ofabout 0.0001% to about 30%.
 19. The medical device of claim 17, whereinthe surface of the medical device contacts a target tissue within thesubject upon use.
 20. The medical device of claim 19, wherein the targettissue is heart tissue or vascular tissue.
 21. The medical device ofclaim 1, wherein the release rate of the anti-connexin agent iscontrolled.
 22. The medical device of claim 1, wherein the medicaldevice provides for surface contact release of the anti-connexin agent.23. The medical device of claim 1, wherein the medical device providesfor the sustained release of the anti-connexin agent.
 24. The medicaldevice of claim 1, wherein the medical device provides for the slowrelease of the anti-connexin agent.
 25. The medical device of claim 1,wherein the device comprises a coating containing an anti-connexinagent.
 26. The medical device of claim 25, wherein the coating comprisesa polymer.
 27. The medical device of claim 25, wherein the coatingcomprises a plurality of layers of a polymer/anti-connexin agentmixture.
 28. The medical device of claim 1, wherein the medical deviceis for implantation in a human.
 29. The medical device of claim 1,wherein the medical device comprises a stent.
 30. The medical device ofclaim 1, wherein the medical device comprises a suture.
 31. The medicaldevice of claim 1, wherein the medical device comprises a balloon, aprosthetic heart valve, an annuloplasty ring, a pulse generator, acardiac defibrillator, an arteriovenous shunt, an anastomosis device, ahemostatic barrier or a pacemaker.
 32. The medical device of claim 1,wherein the medical device comprises an orbital implant, a lens, a lensimplant, or a corneal implant.
 33. The medical device of claim 1,wherein the medical device comprises an orthopedic plate, a bone pin, abone substitute, a anchor, a joint, a screw, or a vertebral disk. 34.The medical device of claim 1, wherein the medical device comprises agraft, a shunt, a vascular implant, a tissue scaffold, an intraluminaldevice or a vascular support.
 35. The medical device of any of claim29-33 or 34, wherein the medical device is for implantation in a human.36. The medical device of claim 1, wherein the medical device comprisesat least one channel formed in an outer surface thereof, and wherein theanti-connexin agent is included on and/or within at least one channel.37. The medical device of claim 1, wherein at least a portion of themedical device is formed, in whole or in part, of a substance thatincludes the anti-connexin agent.
 38. The medical device of claim 1,wherein the device further comprises one or more therapeutic agents. 39.A method of preventing and/or treating damage associated with the use orimplantation of a medical device in a subject comprising introducinginto the subject a medical device of which at least a portion comprisesan anti-connexin 43 agent, wherein the damage is prevented, amerlioratedand/or delayed.
 40. The method of claim 39 wherein the subject is amammal.
 41. The method of claim 39 wherein the mammal is a human. 42.The method of claim 39 wherein the mammal is selected from the groupconsisting of domestic and pet animals, sports animals, farm animals,and zoo animals.
 43. The method of claim 42 wherein the mammal is ahorse, a dog, or a cat.
 44. The method of claim of claim 39, wherein thesurface contacts a site of injury or potential injury.
 45. A method ofpreventing and/or treating damage associated with the use orimplantation of a medical device in a subject comprising use orimplantation of a medical device which comprises an anti-connexin 43agent that is releasable at its point of contact, wherein the damage isprevented, amerliorated and/or delayed.
 46. A medical device for use orimplantation in a mammal, comprising a first coating containing ananti-connexin 43 polynucleotide and a second coating containing ananti-connexin 43 polypeptide releasable upon insertion of the medicaldevice to or within a subject.
 47. A method of manufacturing a medicaldevice, the improvement comprising coating or impregnating the devicewith one or more anti-connexin 43 agents.